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Uev1A promotes AKT-mediated chemoresistance in breast cancer cells. a , b Effects of UEV1 overexpression on chemoresistance of MDA-MB-231 cells. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. After a 4-h exposure to various doses of chemotherapeutic agents Paclitaxel ( a ) or <t>Doxorubicin</t> ( b ), the cells were cultured for an additional 7 days with drug-free medium containing 10% FBS. Then cells were harvested by trypsinization and stained with trypan blue. Cell viability was assayed by cell number counting using a hematocytometer and an inverted microscope. Each sample was measured in triplicate and repeated 2 times. c , d Dependence of chemoresistance of MDA-MB-231 cells to the AKT pathway. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. With 10 μM LY294002 pretreated for 12 h, cells were exposed to various doses of Paclitaxel ( c ) or Doxorubicin ( d ) which were also in the medium with 10 µM LY294002 for 4 h and then cultured for an additional 7 days with medium containing 10% FBS and 10 µM LY294002. Cell viability assay was as described in a , b . e , f Effects of UEV1 overexpression on chemoresistance of MCF7 cells. Experimental conditions were as described in a , b . g , h Dependence of chemoresistance of MCF7 cells to the AKT pathway. Experimental conditions were as described in c , d . * P
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1) Product Images from "Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway"

Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway

Journal: Cancer Cell International

doi: 10.1186/s12935-019-1050-4

Uev1A promotes AKT-mediated chemoresistance in breast cancer cells. a , b Effects of UEV1 overexpression on chemoresistance of MDA-MB-231 cells. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. After a 4-h exposure to various doses of chemotherapeutic agents Paclitaxel ( a ) or Doxorubicin ( b ), the cells were cultured for an additional 7 days with drug-free medium containing 10% FBS. Then cells were harvested by trypsinization and stained with trypan blue. Cell viability was assayed by cell number counting using a hematocytometer and an inverted microscope. Each sample was measured in triplicate and repeated 2 times. c , d Dependence of chemoresistance of MDA-MB-231 cells to the AKT pathway. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. With 10 μM LY294002 pretreated for 12 h, cells were exposed to various doses of Paclitaxel ( c ) or Doxorubicin ( d ) which were also in the medium with 10 µM LY294002 for 4 h and then cultured for an additional 7 days with medium containing 10% FBS and 10 µM LY294002. Cell viability assay was as described in a , b . e , f Effects of UEV1 overexpression on chemoresistance of MCF7 cells. Experimental conditions were as described in a , b . g , h Dependence of chemoresistance of MCF7 cells to the AKT pathway. Experimental conditions were as described in c , d . * P
Figure Legend Snippet: Uev1A promotes AKT-mediated chemoresistance in breast cancer cells. a , b Effects of UEV1 overexpression on chemoresistance of MDA-MB-231 cells. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. After a 4-h exposure to various doses of chemotherapeutic agents Paclitaxel ( a ) or Doxorubicin ( b ), the cells were cultured for an additional 7 days with drug-free medium containing 10% FBS. Then cells were harvested by trypsinization and stained with trypan blue. Cell viability was assayed by cell number counting using a hematocytometer and an inverted microscope. Each sample was measured in triplicate and repeated 2 times. c , d Dependence of chemoresistance of MDA-MB-231 cells to the AKT pathway. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. With 10 μM LY294002 pretreated for 12 h, cells were exposed to various doses of Paclitaxel ( c ) or Doxorubicin ( d ) which were also in the medium with 10 µM LY294002 for 4 h and then cultured for an additional 7 days with medium containing 10% FBS and 10 µM LY294002. Cell viability assay was as described in a , b . e , f Effects of UEV1 overexpression on chemoresistance of MCF7 cells. Experimental conditions were as described in a , b . g , h Dependence of chemoresistance of MCF7 cells to the AKT pathway. Experimental conditions were as described in c , d . * P

Techniques Used: Over Expression, Multiple Displacement Amplification, Plasmid Preparation, Cell Culture, Staining, Inverted Microscopy, Viability Assay

Uev1A inhibits apoptosis through the AKT pathway in breast cancer cells. a , b UEV1A -overexpressed or vector control MDA-MB-231-TR cells with Paclitaxel (Pacl) ( a ) or Doxorubicin (Doxo) ( b ). c , d UEV1A -overexpressed or vector control MCF7 cells with Paclitaxel (Pacl) ( c ) or Doxorubicin (Doxo) ( d ). Cells were pretreated with (right panel) or without (left panel) 10 µM LY294002 (LY) for 12 h and then exposed to Paclitaxel or Doxorubicin, harvested at different time points and the protein levels of total PARP and cleaved-PARP (C-PARP) were detected by western blot. The ectopic Uev1A was monitored by an anti-HA antibody
Figure Legend Snippet: Uev1A inhibits apoptosis through the AKT pathway in breast cancer cells. a , b UEV1A -overexpressed or vector control MDA-MB-231-TR cells with Paclitaxel (Pacl) ( a ) or Doxorubicin (Doxo) ( b ). c , d UEV1A -overexpressed or vector control MCF7 cells with Paclitaxel (Pacl) ( c ) or Doxorubicin (Doxo) ( d ). Cells were pretreated with (right panel) or without (left panel) 10 µM LY294002 (LY) for 12 h and then exposed to Paclitaxel or Doxorubicin, harvested at different time points and the protein levels of total PARP and cleaved-PARP (C-PARP) were detected by western blot. The ectopic Uev1A was monitored by an anti-HA antibody

Techniques Used: Plasmid Preparation, Multiple Displacement Amplification, Western Blot

2) Product Images from "Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers"

Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers

Journal: Cancer Management and Research

doi: 10.2147/CMAR.S160315

Downregulation of HES1 expression could inhibit Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 shRNA plasmids. Cells transfected with empty pLko.1 vector were used as the control group. ( A ) The knockdown efficiency of HES1 was measured by real-time RT-PCR. ( B ) The cell viability of knockdown cell lines was detected by MTT. ( C ) The knockdown and control cells were treated with Doxorubicin (300 μM) for 24 hours and then stained with Multicaspase/7-AAD for the quantitative measurement by using flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of ( C ). ** P
Figure Legend Snippet: Downregulation of HES1 expression could inhibit Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 shRNA plasmids. Cells transfected with empty pLko.1 vector were used as the control group. ( A ) The knockdown efficiency of HES1 was measured by real-time RT-PCR. ( B ) The cell viability of knockdown cell lines was detected by MTT. ( C ) The knockdown and control cells were treated with Doxorubicin (300 μM) for 24 hours and then stained with Multicaspase/7-AAD for the quantitative measurement by using flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of ( C ). ** P

Techniques Used: Expressing, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Quantitative RT-PCR, MTT Assay, Staining, Flow Cytometry, Cytometry

HES1 interplays with PARP1 to regulate AIF subcellular location. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Immunofluorescence of AIF (green) in the HES1-knockdown and control cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. ( B ) Immunofluorescence of AIF (green) in the HES1-overexpressing and control cells. The picture shows the location of AIF (green) in cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. Abbreviations: AIF, apoptosis-inducing factor; PARP1, poly(ADP-ribose) polymerase-1.
Figure Legend Snippet: HES1 interplays with PARP1 to regulate AIF subcellular location. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Immunofluorescence of AIF (green) in the HES1-knockdown and control cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. ( B ) Immunofluorescence of AIF (green) in the HES1-overexpressing and control cells. The picture shows the location of AIF (green) in cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. Abbreviations: AIF, apoptosis-inducing factor; PARP1, poly(ADP-ribose) polymerase-1.

Techniques Used: Immunofluorescence, Staining

HES1 regulates apoptosis by activating PARP1. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Cell apoptosis of HES1-knockdown cell lines was determined by flow cytometry. ( B ) Bars represent the mean of three independent experiments performed in triplicate of ( A ). ( C ) Cell apoptosis of HES1 overexpressing cell line was determined by flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of (C). ** P
Figure Legend Snippet: HES1 regulates apoptosis by activating PARP1. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Cell apoptosis of HES1-knockdown cell lines was determined by flow cytometry. ( B ) Bars represent the mean of three independent experiments performed in triplicate of ( A ). ( C ) Cell apoptosis of HES1 overexpressing cell line was determined by flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of (C). ** P

Techniques Used: Flow Cytometry, Cytometry

Upregulation of HES1 expression could promote Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 overexpression plasmids. Cells transfected with empty pOZ vector were used as the control group. ( A ) The overexpression efficiency of HES1 was measured by real-time RT-PCR. ( B ) The levels of the overexpressed protein were detected by Western blot. ( C ) The cell viability of overexpressed cell line was detected by MTT. ( D ) The overexpressed cells were stained with Multicaspase/7-AAD for quantitative measurement by using flow cytometry. ( E ) Bars represent the mean of three independent experiments performed in triplicate of ( D ). # P > 0.05, ** P
Figure Legend Snippet: Upregulation of HES1 expression could promote Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 overexpression plasmids. Cells transfected with empty pOZ vector were used as the control group. ( A ) The overexpression efficiency of HES1 was measured by real-time RT-PCR. ( B ) The levels of the overexpressed protein were detected by Western blot. ( C ) The cell viability of overexpressed cell line was detected by MTT. ( D ) The overexpressed cells were stained with Multicaspase/7-AAD for quantitative measurement by using flow cytometry. ( E ) Bars represent the mean of three independent experiments performed in triplicate of ( D ). # P > 0.05, ** P

Techniques Used: Expressing, Stable Transfection, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, MTT Assay, Staining, Flow Cytometry, Cytometry

Doxorubicin affects Hela S3 cell apoptosis. Notes: The HeLa S3 cells were treated with Doxorubicin (0, 100, 300, 600, 900 and 1200 μM) for 24 hours. ( A ) The percentage of apoptosis cells is shown. ( B ) Bars represent mean of three independent experiments performed in triplicate.
Figure Legend Snippet: Doxorubicin affects Hela S3 cell apoptosis. Notes: The HeLa S3 cells were treated with Doxorubicin (0, 100, 300, 600, 900 and 1200 μM) for 24 hours. ( A ) The percentage of apoptosis cells is shown. ( B ) Bars represent mean of three independent experiments performed in triplicate.

Techniques Used:

Effects of different concentrations of Doxorubicin on the mRNA expression of Notch pathway members. Notes: ( A ) HeLa S3 cells were treated with Doxorubicin (0, 300, 900 and 1200 μM) for 24 hours. The mRNA expression levels of Notch pathway components and targets were examined by real-time RT-PCR. ( B ) Selective representation of Doxorubicin-responsive Notch pathway genes. GAPDH was used as an internal control for the normalization of gene expression. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.
Figure Legend Snippet: Effects of different concentrations of Doxorubicin on the mRNA expression of Notch pathway members. Notes: ( A ) HeLa S3 cells were treated with Doxorubicin (0, 300, 900 and 1200 μM) for 24 hours. The mRNA expression levels of Notch pathway components and targets were examined by real-time RT-PCR. ( B ) Selective representation of Doxorubicin-responsive Notch pathway genes. GAPDH was used as an internal control for the normalization of gene expression. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.

Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

3) Product Images from "A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission"

Article Title: A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission

Journal: Autophagy

doi: 10.1080/15548627.2015.1056970

Pharmacological or genetic inhibition of DNM1L blocks mitochondrial fission and apoptosis induced by liensinine in combination with doxorubicin. ( A ) MDA-MB-231 cells were co-exposed to Lien (20 μM) and Dox (0.2 μM) in the presence or absence
Figure Legend Snippet: Pharmacological or genetic inhibition of DNM1L blocks mitochondrial fission and apoptosis induced by liensinine in combination with doxorubicin. ( A ) MDA-MB-231 cells were co-exposed to Lien (20 μM) and Dox (0.2 μM) in the presence or absence

Techniques Used: Polyacrylamide Gel Electrophoresis, Inhibition, Multiple Displacement Amplification

The combination of liensinine and doxorubicin inhibits tumor growth in a MDA-MB-231 mouse xenograft model. ( A ) Average tumor volume in vehicle control mice, mice treated with Lien (60 mg/kg) or Dox (2 mg/kg) or a combination of Lien and
Figure Legend Snippet: The combination of liensinine and doxorubicin inhibits tumor growth in a MDA-MB-231 mouse xenograft model. ( A ) Average tumor volume in vehicle control mice, mice treated with Lien (60 mg/kg) or Dox (2 mg/kg) or a combination of Lien and

Techniques Used: Multiple Displacement Amplification, Mouse Assay

Excessive accumulation of autophagosomes/mitophagosomes mediated by combination of liensinine and doxorubicin in MDA-MB-231 cells. ( A ) Cells were treated with vehicle, Lien (20 μM), Dox (0.2 μM) alone or combined treated
Figure Legend Snippet: Excessive accumulation of autophagosomes/mitophagosomes mediated by combination of liensinine and doxorubicin in MDA-MB-231 cells. ( A ) Cells were treated with vehicle, Lien (20 μM), Dox (0.2 μM) alone or combined treated

Techniques Used: Multiple Displacement Amplification

Autophagic inhibition combined with doxorubicin induces mitochondrial fission via dephosphorylation and mitochondrial translocation of DNM1L. ( A and D ) MDA-MB-231 cells were treated with vehicle, Lien (20 μM), Dox (0.2 μM)
Figure Legend Snippet: Autophagic inhibition combined with doxorubicin induces mitochondrial fission via dephosphorylation and mitochondrial translocation of DNM1L. ( A and D ) MDA-MB-231 cells were treated with vehicle, Lien (20 μM), Dox (0.2 μM)

Techniques Used: Inhibition, De-Phosphorylation Assay, Translocation Assay, Multiple Displacement Amplification

Liensinine sensitizes to a variety of anticancer drug-induced cell death in MDA-MB-231 and MCF-7 cells. ( A ) MDA-MB-231 and MCF-7 cells were treated with various concentrations of doxorubicin (Dox), paclitaxel, vincristine, cisplatin (CDDP), and staurosporine
Figure Legend Snippet: Liensinine sensitizes to a variety of anticancer drug-induced cell death in MDA-MB-231 and MCF-7 cells. ( A ) MDA-MB-231 and MCF-7 cells were treated with various concentrations of doxorubicin (Dox), paclitaxel, vincristine, cisplatin (CDDP), and staurosporine

Techniques Used: Multiple Displacement Amplification

Autophagosome/mitophagosome accumulation contributed to the combination effect of liensinine and doxorubicin. MDA-MB-231 cells were co-exposed to Lien (20 μM) and Dox (0.2 μM) in the presence or absence of 3-MA (5 mM)
Figure Legend Snippet: Autophagosome/mitophagosome accumulation contributed to the combination effect of liensinine and doxorubicin. MDA-MB-231 cells were co-exposed to Lien (20 μM) and Dox (0.2 μM) in the presence or absence of 3-MA (5 mM)

Techniques Used: Polyacrylamide Gel Electrophoresis, Multiple Displacement Amplification

4) Product Images from "Anti-inflammatory and Antitumor Activity of a Triple Therapy for a Colitis-Related Colorectal Cancer"

Article Title: Anti-inflammatory and Antitumor Activity of a Triple Therapy for a Colitis-Related Colorectal Cancer

Journal: Journal of Cancer

doi: 10.7150/jca.13123

Metformin - Sodium Oxamate - Doxorubicin increased apoptosis cell death. It is shown a significant increment in the detection of caspase 3 and PARP-1 after exposure to the three drugs in combination. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin.
Figure Legend Snippet: Metformin - Sodium Oxamate - Doxorubicin increased apoptosis cell death. It is shown a significant increment in the detection of caspase 3 and PARP-1 after exposure to the three drugs in combination. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin.

Techniques Used:

Combinatorial therapy induced glycolysis inhibition . The suppression of LDH-A enzyme since 4 h to drugs exposure revels the inhibition of the glycolysis pathway. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin.
Figure Legend Snippet: Combinatorial therapy induced glycolysis inhibition . The suppression of LDH-A enzyme since 4 h to drugs exposure revels the inhibition of the glycolysis pathway. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin.

Techniques Used: Inhibition

Histological and Immunohistochemical Analysis of the large bowel during the progression of the model and experimental treatments. Samples were collected at the 6 th day during the resting period after each DSS dose followed by paraffin inclusion and H E staining. A, Healthy tissue where m=muscle and c=crypts; B, DSS-1; C, DSS-2; D, SSI or Colorectal Cancer; E, Doxorubicin 1 mg/Kg; F, Metformin - Sodium Oxamate (200-15) mg/Kg and G, Metformin - Sodium Oxamate - Doxorubicin (200-15-1) mg/Kg. 100X magnification. During the curse of the model, arrows shown slight crypt distortion, branched crypts in cross section as well as epithelial hyperplasia, reduction in globet cell numbers until adenoma with intramucosal carcinoma and haphazard proliferation of malignant cells within the lamina propria.
Figure Legend Snippet: Histological and Immunohistochemical Analysis of the large bowel during the progression of the model and experimental treatments. Samples were collected at the 6 th day during the resting period after each DSS dose followed by paraffin inclusion and H E staining. A, Healthy tissue where m=muscle and c=crypts; B, DSS-1; C, DSS-2; D, SSI or Colorectal Cancer; E, Doxorubicin 1 mg/Kg; F, Metformin - Sodium Oxamate (200-15) mg/Kg and G, Metformin - Sodium Oxamate - Doxorubicin (200-15-1) mg/Kg. 100X magnification. During the curse of the model, arrows shown slight crypt distortion, branched crypts in cross section as well as epithelial hyperplasia, reduction in globet cell numbers until adenoma with intramucosal carcinoma and haphazard proliferation of malignant cells within the lamina propria.

Techniques Used: Immunohistochemistry, Staining

Levels of protein cytokines in the experimental groups . Samples were collected at the 6 th day of the rest period after each DSS dose followed by RIPA protein and TRIZOL RNA extractions then, Bio-Plex mouse Cytokine eight-plex Assay and qRT-PCR respectively. A, IL-1β pro-inflammatory and IL-10 an anti-inflammatory cytokine. B, TNFα, IL-6, INFγ, IL-4 and IL-10 relative expression compared with the control healthy group. Each bar represents the mean ± SD from three independent experiments. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin and SSI: Sodium Saline Isotonic.
Figure Legend Snippet: Levels of protein cytokines in the experimental groups . Samples were collected at the 6 th day of the rest period after each DSS dose followed by RIPA protein and TRIZOL RNA extractions then, Bio-Plex mouse Cytokine eight-plex Assay and qRT-PCR respectively. A, IL-1β pro-inflammatory and IL-10 an anti-inflammatory cytokine. B, TNFα, IL-6, INFγ, IL-4 and IL-10 relative expression compared with the control healthy group. Each bar represents the mean ± SD from three independent experiments. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin and SSI: Sodium Saline Isotonic.

Techniques Used: Plex Assay, Quantitative RT-PCR, Expressing

Induction of autophagy through the combinatorial therapy. Metformin - Sodium Oxamate - Doxorubicin induced Autophagy, denoted as LC3 conversion (LC3-II) and abscence of p-mTOR from 4 h on to drug exposure. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin.
Figure Legend Snippet: Induction of autophagy through the combinatorial therapy. Metformin - Sodium Oxamate - Doxorubicin induced Autophagy, denoted as LC3 conversion (LC3-II) and abscence of p-mTOR from 4 h on to drug exposure. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin.

Techniques Used:

Met-Ox-Dox treatment inhibits growth of colorectal cancer in an in vivo murine model . A, experimental strategy of the model with days of exposure to Azoxymethane (AOM) and dextran sulfate sodium (DSS), scheme of treatment with the pharmacological proposed therapy; B, C and D, Mice weight monitoring during the experimental plan, average of number and size of tumors after macroscopic inspection of the large bowels at the end of treatments; and E, macroscopic view of the large bowels at the end of the treatment. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin and SSI: Sodium Saline Isotonic. Significance of * p
Figure Legend Snippet: Met-Ox-Dox treatment inhibits growth of colorectal cancer in an in vivo murine model . A, experimental strategy of the model with days of exposure to Azoxymethane (AOM) and dextran sulfate sodium (DSS), scheme of treatment with the pharmacological proposed therapy; B, C and D, Mice weight monitoring during the experimental plan, average of number and size of tumors after macroscopic inspection of the large bowels at the end of treatments; and E, macroscopic view of the large bowels at the end of the treatment. Met: Metformin; Ox: Sodium Oxamate; Dox: Doxorubicin and SSI: Sodium Saline Isotonic. Significance of * p

Techniques Used: In Vivo, Mouse Assay

5) Product Images from "Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses"

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2019.01.003

Transduction of Primary Human Cells with rAAV/BoV Vectors (A) Heatmap showing the results of transduction of the indicated primary cells with the different scAAV-Gluc/BoV vectors at an MOI of 5 × 10 4 . Plotted is the Gluc activity measured in the medium 9 days post-transduction. (B) Transduction of primary myotubes with 1 × 10 9 genomes of scAAV-YFP/BoV vectors or AAV2 as control. (C and D) Transduction of differentiated primary human colon organoids with 5 × 10 9 viral genomes of scAAV-Gluc/BoV (C) or 1 × 10 11 viral genomes of scAAV-YFP/BoV (D). Gluc activity shown in (C) was measured at the indicated time points. Data represent the mean and range of two independent experiments (n = 2 donors). Images in (D) were taken 9 days post-transduction. The exposure was reduced 3-fold for AAV2 to avoid saturation of signal. All transductions were performed in the presence of 1 μM Doxorubicin. For all images, cells were fixed and stained with an FITC-coupled anti-GFP antibody. Scale bar, 50 μm. Nuclei were stained with Hoechst. SK, skeletal muscle cells; cardio, cardiac myocytes; pul, pulmonary fibroblasts; vein, saphenous vein endothelial cells. Dashed lines indicate the assay background.
Figure Legend Snippet: Transduction of Primary Human Cells with rAAV/BoV Vectors (A) Heatmap showing the results of transduction of the indicated primary cells with the different scAAV-Gluc/BoV vectors at an MOI of 5 × 10 4 . Plotted is the Gluc activity measured in the medium 9 days post-transduction. (B) Transduction of primary myotubes with 1 × 10 9 genomes of scAAV-YFP/BoV vectors or AAV2 as control. (C and D) Transduction of differentiated primary human colon organoids with 5 × 10 9 viral genomes of scAAV-Gluc/BoV (C) or 1 × 10 11 viral genomes of scAAV-YFP/BoV (D). Gluc activity shown in (C) was measured at the indicated time points. Data represent the mean and range of two independent experiments (n = 2 donors). Images in (D) were taken 9 days post-transduction. The exposure was reduced 3-fold for AAV2 to avoid saturation of signal. All transductions were performed in the presence of 1 μM Doxorubicin. For all images, cells were fixed and stained with an FITC-coupled anti-GFP antibody. Scale bar, 50 μm. Nuclei were stained with Hoechst. SK, skeletal muscle cells; cardio, cardiac myocytes; pul, pulmonary fibroblasts; vein, saphenous vein endothelial cells. Dashed lines indicate the assay background.

Techniques Used: Transduction, Activity Assay, Staining

Pseudotyping of rAAV Genomes with Capsids Derived from Four Additional Bocavirus Serotypes (A) BoV helper plasmid (pCMVNS*Cap1) for chimeric rAAV/HBoV1 production and acceptor plasmid (pCMVNS*CapΔ) derived thereof for cloning of the different BoV cap ORFs. Each cap sequence was ordered as two gene blocks, assembled to a full-length cap ORF (cap x , where x = HBoV2–4 or GBoV) and subsequently cloned into the acceptor plasmid using a Golden Gate reaction. BocaSR, BoV-transcribed small non-coding RNA. Numbers in brackets refer to the construct labels in Figure 1 A. (B) Production and iodixanol purification of chimeric HBoV1-4 and GBoV vectors encoding Gluc. The amount of genome copies per milliliter was determined with TaqMan RT-PCR. Shown are averages (±SEM) of four independent productions. (C) Western blot analysis of the indicated iodixanol-purified BoV stocks. Detected are the three BoV capsid proteins VP1, VP2, and VP3. NEG, iodixanol gradient from untransfected cells. (D) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 2 × 10 4 . Gluc activity in the medium was measured at 4 and 9 days post-transduction as arbitrary light units (ALU). Data are the mean Gluc expression (±SEM, n = 3). Numbers above the columns depict fold increases in expression. (E) Flow cytometry analysis of pHAEs transduced with scAAV-YFP/HBoV1 or scAAV-YFP/HBoV4. scAAV-Gluc/HBoV1 was used as a control (both at an MOI of 5 × 10 4 , n = 4 independent transwells). Cells were co-stained for YFP and a cell type-specific marker: β-Tubulin IV (ciliated cells), MUC5AC (goblet cells), CC10 (club cells), or KRT5 (basal cells). Percentages of double-positive cells are shown in the upper right quarter. (F) Transduction of primary lung organoids with the indicated scAAV-Gluc/BoV variants. Several organoids per donor were either mechanically broken (br) and incubated with a total of 5 × 10 9 viral genomes, or they were individually microinjected (in) with 5 × 10 8 –1 × 10 9 viral particles. Total Gluc activity in the medium was measured 3–12 days post-transduction and plotted on the y axis as ALU. Numbers above the columns depict fold increases in expression. (G) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 1 × 10 4 in the presence (+) or absence (−) of IVIg. Gluc activity in the medium was measured 5 days post-transduction as ALU. Shown is the mean Gluc activity (+SEM, n = 4, except for HB1 [–] n = 3). For pHAEs transduction, LLnL and Doxorubicin were added at concentrations of 40 and 5 μM, respectively. Transductions of primary lung organoids were performed in the presence of 1 μM Doxorubicin. For statistical analysis, one-way ANOVA was used. Significance at p
Figure Legend Snippet: Pseudotyping of rAAV Genomes with Capsids Derived from Four Additional Bocavirus Serotypes (A) BoV helper plasmid (pCMVNS*Cap1) for chimeric rAAV/HBoV1 production and acceptor plasmid (pCMVNS*CapΔ) derived thereof for cloning of the different BoV cap ORFs. Each cap sequence was ordered as two gene blocks, assembled to a full-length cap ORF (cap x , where x = HBoV2–4 or GBoV) and subsequently cloned into the acceptor plasmid using a Golden Gate reaction. BocaSR, BoV-transcribed small non-coding RNA. Numbers in brackets refer to the construct labels in Figure 1 A. (B) Production and iodixanol purification of chimeric HBoV1-4 and GBoV vectors encoding Gluc. The amount of genome copies per milliliter was determined with TaqMan RT-PCR. Shown are averages (±SEM) of four independent productions. (C) Western blot analysis of the indicated iodixanol-purified BoV stocks. Detected are the three BoV capsid proteins VP1, VP2, and VP3. NEG, iodixanol gradient from untransfected cells. (D) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 2 × 10 4 . Gluc activity in the medium was measured at 4 and 9 days post-transduction as arbitrary light units (ALU). Data are the mean Gluc expression (±SEM, n = 3). Numbers above the columns depict fold increases in expression. (E) Flow cytometry analysis of pHAEs transduced with scAAV-YFP/HBoV1 or scAAV-YFP/HBoV4. scAAV-Gluc/HBoV1 was used as a control (both at an MOI of 5 × 10 4 , n = 4 independent transwells). Cells were co-stained for YFP and a cell type-specific marker: β-Tubulin IV (ciliated cells), MUC5AC (goblet cells), CC10 (club cells), or KRT5 (basal cells). Percentages of double-positive cells are shown in the upper right quarter. (F) Transduction of primary lung organoids with the indicated scAAV-Gluc/BoV variants. Several organoids per donor were either mechanically broken (br) and incubated with a total of 5 × 10 9 viral genomes, or they were individually microinjected (in) with 5 × 10 8 –1 × 10 9 viral particles. Total Gluc activity in the medium was measured 3–12 days post-transduction and plotted on the y axis as ALU. Numbers above the columns depict fold increases in expression. (G) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 1 × 10 4 in the presence (+) or absence (−) of IVIg. Gluc activity in the medium was measured 5 days post-transduction as ALU. Shown is the mean Gluc activity (+SEM, n = 4, except for HB1 [–] n = 3). For pHAEs transduction, LLnL and Doxorubicin were added at concentrations of 40 and 5 μM, respectively. Transductions of primary lung organoids were performed in the presence of 1 μM Doxorubicin. For statistical analysis, one-way ANOVA was used. Significance at p

Techniques Used: Derivative Assay, Plasmid Preparation, Clone Assay, Sequencing, Construct, Purification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transduction, Activity Assay, Expressing, Flow Cytometry, Cytometry, Staining, Marker, Incubation

Transduction of Primary Human Hepatocytes and T Cells with rAAV/BoV Vectors (A) YFP expression 5 days after transduction of primary human hepatocytes (pHeps) with scAAV-YFP/BoV vectors at the indicated MOIs (n = 3 donors). Expression first became detectable at day 2 (not shown) and then increased over time. Cells were fixed and stained with an FITC-coupled anti-GFP antibody, and nuclei were stained with Hoechst. Scale bar, 50 μm. (B) Gluc activity at 3 and 6 days post-transduction of pHeps with scAAV-Gluc/BoV at an MOI of 1 × 10 4 . Data represent the mean and range of two independent measurements (n = 2 donors). (C) Transduction of primary human T cells with scAAV-Gluc/BoV (MOI = 1 × 10 4 ). Gluc activity in the medium was measured 3–12 days post-transduction. Plotted are means ±SEM of three independent experiments (n = 3 donors). For all transductions, Doxorubicin was added to a final concentration of 1 μM/well. Dashed lines indicate the assay background.
Figure Legend Snippet: Transduction of Primary Human Hepatocytes and T Cells with rAAV/BoV Vectors (A) YFP expression 5 days after transduction of primary human hepatocytes (pHeps) with scAAV-YFP/BoV vectors at the indicated MOIs (n = 3 donors). Expression first became detectable at day 2 (not shown) and then increased over time. Cells were fixed and stained with an FITC-coupled anti-GFP antibody, and nuclei were stained with Hoechst. Scale bar, 50 μm. (B) Gluc activity at 3 and 6 days post-transduction of pHeps with scAAV-Gluc/BoV at an MOI of 1 × 10 4 . Data represent the mean and range of two independent measurements (n = 2 donors). (C) Transduction of primary human T cells with scAAV-Gluc/BoV (MOI = 1 × 10 4 ). Gluc activity in the medium was measured 3–12 days post-transduction. Plotted are means ±SEM of three independent experiments (n = 3 donors). For all transductions, Doxorubicin was added to a final concentration of 1 μM/well. Dashed lines indicate the assay background.

Techniques Used: Transduction, Expressing, Staining, Activity Assay, Concentration Assay

6) Product Images from "Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers"

Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers

Journal: Cancer Management and Research

doi: 10.2147/CMAR.S160315

Downregulation of HES1 expression could inhibit Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 shRNA plasmids. Cells transfected with empty pLko.1 vector were used as the control group. ( A ) The knockdown efficiency of HES1 was measured by real-time RT-PCR. ( B ) The cell viability of knockdown cell lines was detected by MTT. ( C ) The knockdown and control cells were treated with Doxorubicin (300 μM) for 24 hours and then stained with Multicaspase/7-AAD for the quantitative measurement by using flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of ( C ). ** P
Figure Legend Snippet: Downregulation of HES1 expression could inhibit Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 shRNA plasmids. Cells transfected with empty pLko.1 vector were used as the control group. ( A ) The knockdown efficiency of HES1 was measured by real-time RT-PCR. ( B ) The cell viability of knockdown cell lines was detected by MTT. ( C ) The knockdown and control cells were treated with Doxorubicin (300 μM) for 24 hours and then stained with Multicaspase/7-AAD for the quantitative measurement by using flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of ( C ). ** P

Techniques Used: Expressing, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Quantitative RT-PCR, MTT Assay, Staining, Flow Cytometry, Cytometry

HES1 interplays with PARP1 to regulate AIF subcellular location. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Immunofluorescence of AIF (green) in the HES1-knockdown and control cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. ( B ) Immunofluorescence of AIF (green) in the HES1-overexpressing and control cells. The picture shows the location of AIF (green) in cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. Abbreviations: AIF, apoptosis-inducing factor; PARP1, poly(ADP-ribose) polymerase-1.
Figure Legend Snippet: HES1 interplays with PARP1 to regulate AIF subcellular location. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Immunofluorescence of AIF (green) in the HES1-knockdown and control cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. ( B ) Immunofluorescence of AIF (green) in the HES1-overexpressing and control cells. The picture shows the location of AIF (green) in cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. Abbreviations: AIF, apoptosis-inducing factor; PARP1, poly(ADP-ribose) polymerase-1.

Techniques Used: Immunofluorescence, Staining

HES1 regulates apoptosis by activating PARP1. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Cell apoptosis of HES1-knockdown cell lines was determined by flow cytometry. ( B ) Bars represent the mean of three independent experiments performed in triplicate of ( A ). ( C ) Cell apoptosis of HES1 overexpressing cell line was determined by flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of (C). ** P
Figure Legend Snippet: HES1 regulates apoptosis by activating PARP1. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Cell apoptosis of HES1-knockdown cell lines was determined by flow cytometry. ( B ) Bars represent the mean of three independent experiments performed in triplicate of ( A ). ( C ) Cell apoptosis of HES1 overexpressing cell line was determined by flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of (C). ** P

Techniques Used: Flow Cytometry, Cytometry

Upregulation of HES1 expression could promote Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 overexpression plasmids. Cells transfected with empty pOZ vector were used as the control group. ( A ) The overexpression efficiency of HES1 was measured by real-time RT-PCR. ( B ) The levels of the overexpressed protein were detected by Western blot. ( C ) The cell viability of overexpressed cell line was detected by MTT. ( D ) The overexpressed cells were stained with Multicaspase/7-AAD for quantitative measurement by using flow cytometry. ( E ) Bars represent the mean of three independent experiments performed in triplicate of ( D ). # P > 0.05, ** P
Figure Legend Snippet: Upregulation of HES1 expression could promote Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 overexpression plasmids. Cells transfected with empty pOZ vector were used as the control group. ( A ) The overexpression efficiency of HES1 was measured by real-time RT-PCR. ( B ) The levels of the overexpressed protein were detected by Western blot. ( C ) The cell viability of overexpressed cell line was detected by MTT. ( D ) The overexpressed cells were stained with Multicaspase/7-AAD for quantitative measurement by using flow cytometry. ( E ) Bars represent the mean of three independent experiments performed in triplicate of ( D ). # P > 0.05, ** P

Techniques Used: Expressing, Stable Transfection, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, MTT Assay, Staining, Flow Cytometry, Cytometry

Doxorubicin affects Hela S3 cell apoptosis. Notes: The HeLa S3 cells were treated with Doxorubicin (0, 100, 300, 600, 900 and 1200 μM) for 24 hours. ( A ) The percentage of apoptosis cells is shown. ( B ) Bars represent mean of three independent experiments performed in triplicate.
Figure Legend Snippet: Doxorubicin affects Hela S3 cell apoptosis. Notes: The HeLa S3 cells were treated with Doxorubicin (0, 100, 300, 600, 900 and 1200 μM) for 24 hours. ( A ) The percentage of apoptosis cells is shown. ( B ) Bars represent mean of three independent experiments performed in triplicate.

Techniques Used:

Effects of different concentrations of Doxorubicin on the mRNA expression of Notch pathway members. Notes: ( A ) HeLa S3 cells were treated with Doxorubicin (0, 300, 900 and 1200 μM) for 24 hours. The mRNA expression levels of Notch pathway components and targets were examined by real-time RT-PCR. ( B ) Selective representation of Doxorubicin-responsive Notch pathway genes. GAPDH was used as an internal control for the normalization of gene expression. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.
Figure Legend Snippet: Effects of different concentrations of Doxorubicin on the mRNA expression of Notch pathway members. Notes: ( A ) HeLa S3 cells were treated with Doxorubicin (0, 300, 900 and 1200 μM) for 24 hours. The mRNA expression levels of Notch pathway components and targets were examined by real-time RT-PCR. ( B ) Selective representation of Doxorubicin-responsive Notch pathway genes. GAPDH was used as an internal control for the normalization of gene expression. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.

Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

7) Product Images from "Phosphorylation-Dependent Degradation of p300 by Doxorubicin-Activated p38 Mitogen-Activated Protein Kinase in Cardiac Cells"

Article Title: Phosphorylation-Dependent Degradation of p300 by Doxorubicin-Activated p38 Mitogen-Activated Protein Kinase in Cardiac Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.7.2673-2687.2005

Suppression of doxorubicin-induced p300 degradation by inhibition of p38 MAPK. (A) Primary cardiomyocytes were treated with various concentrations of SB 202190, and p300 protein levels were determined by Western blot analysis with an anti-p300 antibody.
Figure Legend Snippet: Suppression of doxorubicin-induced p300 degradation by inhibition of p38 MAPK. (A) Primary cardiomyocytes were treated with various concentrations of SB 202190, and p300 protein levels were determined by Western blot analysis with an anti-p300 antibody.

Techniques Used: Inhibition, Western Blot

Doxorubicin induces phosphorylation of p300 in cardiomyocytes in culture, and inhibition of the proteasome activity reduces degradation of phosphorylated p300. (A) Primary cardiomyocytes were maintained in medium without phosphate and then labeled with
Figure Legend Snippet: Doxorubicin induces phosphorylation of p300 in cardiomyocytes in culture, and inhibition of the proteasome activity reduces degradation of phosphorylated p300. (A) Primary cardiomyocytes were maintained in medium without phosphate and then labeled with

Techniques Used: Inhibition, Activity Assay, Labeling

p300 degradation in cardiomyocytes treated with doxorubicin parallels apoptosis. (A) Primary neonatal cardiomyocytes plated on coverslides were maintained in normal medium (Untr.) or in medium supplemented with 1 μM doxorubicin for 24 h (Dox 24
Figure Legend Snippet: p300 degradation in cardiomyocytes treated with doxorubicin parallels apoptosis. (A) Primary neonatal cardiomyocytes plated on coverslides were maintained in normal medium (Untr.) or in medium supplemented with 1 μM doxorubicin for 24 h (Dox 24

Techniques Used:

p300 degradation and stabilization of the p53 protein after doxorubicin treatment. Primary neonatal cardiomyocytes plated on coverslides were maintained in normal medium (control) or in medium supplemented with 1 μM doxorubicin for 24 h (Dox 24
Figure Legend Snippet: p300 degradation and stabilization of the p53 protein after doxorubicin treatment. Primary neonatal cardiomyocytes plated on coverslides were maintained in normal medium (control) or in medium supplemented with 1 μM doxorubicin for 24 h (Dox 24

Techniques Used:

Loss of GATA-4 following doxorubicin exposure is not mediated by depletion of p300 protein. Primary neonatal rat cardiomyocytes were isolated and maintained in normal medium in medium supplemented with 1 μM doxorubicin (Dox), or in medium containing
Figure Legend Snippet: Loss of GATA-4 following doxorubicin exposure is not mediated by depletion of p300 protein. Primary neonatal rat cardiomyocytes were isolated and maintained in normal medium in medium supplemented with 1 μM doxorubicin (Dox), or in medium containing

Techniques Used: Isolation

Model of p38-mediated degradation of p300. On treatment with doxorubicin (Dox), neonatal cardiomyocytes up-regulate the activity of p38, which targets the N-terminal and C-terminal regions of p300 for phosphorylation. Phosphorylated p300 is then targeted
Figure Legend Snippet: Model of p38-mediated degradation of p300. On treatment with doxorubicin (Dox), neonatal cardiomyocytes up-regulate the activity of p38, which targets the N-terminal and C-terminal regions of p300 for phosphorylation. Phosphorylated p300 is then targeted

Techniques Used: Activity Assay

A serine/threonine inhibitor stabilizes the p300 protein level, and doxorubicin (Dox) treatment induces threonine phosphorylation of p300 in cardiomyocytes. (A) Primary neonatal cardiomyocytes were treated with 1 nM staurosporine or 3 μM H-7 and
Figure Legend Snippet: A serine/threonine inhibitor stabilizes the p300 protein level, and doxorubicin (Dox) treatment induces threonine phosphorylation of p300 in cardiomyocytes. (A) Primary neonatal cardiomyocytes were treated with 1 nM staurosporine or 3 μM H-7 and

Techniques Used:

Doxorubicin activates pp38 in cardiomyocytes. Primary neonatal cardiomyocytes were plated on coverslides and maintained in normal medium (Untr.) or treated with 1 μM doxorubicin for 12 or 24 h (Dox 12 h, Dox 24 h). (A and B) The cells were incubated
Figure Legend Snippet: Doxorubicin activates pp38 in cardiomyocytes. Primary neonatal cardiomyocytes were plated on coverslides and maintained in normal medium (Untr.) or treated with 1 μM doxorubicin for 12 or 24 h (Dox 12 h, Dox 24 h). (A and B) The cells were incubated

Techniques Used: Incubation

Interaction of p300 and p38 in cardiomyocytes in vivo. Primary neonatal cardiomyocytes were maintained for 12 h in normal medium or in medium supplemented with 1 μM doxorubicin (Dox). Nuclear extracts were prepared, and equal amounts of nuclear
Figure Legend Snippet: Interaction of p300 and p38 in cardiomyocytes in vivo. Primary neonatal cardiomyocytes were maintained for 12 h in normal medium or in medium supplemented with 1 μM doxorubicin (Dox). Nuclear extracts were prepared, and equal amounts of nuclear

Techniques Used: In Vivo

8) Product Images from "Bcl-xL mediates therapeutic resistance of a mesenchymal breast cancer cell subpopulation"

Article Title: Bcl-xL mediates therapeutic resistance of a mesenchymal breast cancer cell subpopulation

Journal: Oncotarget

doi:

MSP RAS cells resist chemotherapeutic treatment and apoptosis induction (A) HMLE RAS cells were treated with 5μM or 10μM cisplatin, for 24h, followed by further incubation without chemotherapeutics. The cell number was determined daily for 7d, using automated light microscopy with quantitative image analysis (Celigo cytometer). Non-treated cells were used as controls. (B-F) HMLE RAS cells were treated with the indicated concentrations of cisplatin (B) or carboplatin (C), each for 16h, with doxorubicin for 24h (D), with neocarzinostatin for 20h (E) or Trail in the presence of 20μg/ml cycloheximide for 6h (F). Controls were treated with corresponding amounts of the respective solvent. Cell lysates were analysed by immunoblotting. β-Actin or HSC70 staining was used as loading control.
Figure Legend Snippet: MSP RAS cells resist chemotherapeutic treatment and apoptosis induction (A) HMLE RAS cells were treated with 5μM or 10μM cisplatin, for 24h, followed by further incubation without chemotherapeutics. The cell number was determined daily for 7d, using automated light microscopy with quantitative image analysis (Celigo cytometer). Non-treated cells were used as controls. (B-F) HMLE RAS cells were treated with the indicated concentrations of cisplatin (B) or carboplatin (C), each for 16h, with doxorubicin for 24h (D), with neocarzinostatin for 20h (E) or Trail in the presence of 20μg/ml cycloheximide for 6h (F). Controls were treated with corresponding amounts of the respective solvent. Cell lysates were analysed by immunoblotting. β-Actin or HSC70 staining was used as loading control.

Techniques Used: Incubation, Light Microscopy, Cytometry, Staining

9) Product Images from "Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses"

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2019.01.003

Transduction of Primary Human Cells with rAAV/BoV Vectors (A) Heatmap showing the results of transduction of the indicated primary cells with the different scAAV-Gluc/BoV vectors at an MOI of 5 × 10 4 . Plotted is the Gluc activity measured in the medium 9 days post-transduction. (B) Transduction of primary myotubes with 1 × 10 9 genomes of scAAV-YFP/BoV vectors or AAV2 as control. (C and D) Transduction of differentiated primary human colon organoids with 5 × 10 9 viral genomes of scAAV-Gluc/BoV (C) or 1 × 10 11 viral genomes of scAAV-YFP/BoV (D). Gluc activity shown in (C) was measured at the indicated time points. Data represent the mean and range of two independent experiments (n = 2 donors). Images in (D) were taken 9 days post-transduction. The exposure was reduced 3-fold for AAV2 to avoid saturation of signal. All transductions were performed in the presence of 1 μM Doxorubicin. For all images, cells were fixed and stained with an FITC-coupled anti-GFP antibody. Scale bar, 50 μm. Nuclei were stained with Hoechst. SK, skeletal muscle cells; cardio, cardiac myocytes; pul, pulmonary fibroblasts; vein, saphenous vein endothelial cells. Dashed lines indicate the assay background.
Figure Legend Snippet: Transduction of Primary Human Cells with rAAV/BoV Vectors (A) Heatmap showing the results of transduction of the indicated primary cells with the different scAAV-Gluc/BoV vectors at an MOI of 5 × 10 4 . Plotted is the Gluc activity measured in the medium 9 days post-transduction. (B) Transduction of primary myotubes with 1 × 10 9 genomes of scAAV-YFP/BoV vectors or AAV2 as control. (C and D) Transduction of differentiated primary human colon organoids with 5 × 10 9 viral genomes of scAAV-Gluc/BoV (C) or 1 × 10 11 viral genomes of scAAV-YFP/BoV (D). Gluc activity shown in (C) was measured at the indicated time points. Data represent the mean and range of two independent experiments (n = 2 donors). Images in (D) were taken 9 days post-transduction. The exposure was reduced 3-fold for AAV2 to avoid saturation of signal. All transductions were performed in the presence of 1 μM Doxorubicin. For all images, cells were fixed and stained with an FITC-coupled anti-GFP antibody. Scale bar, 50 μm. Nuclei were stained with Hoechst. SK, skeletal muscle cells; cardio, cardiac myocytes; pul, pulmonary fibroblasts; vein, saphenous vein endothelial cells. Dashed lines indicate the assay background.

Techniques Used: Transduction, Activity Assay, Staining

Pseudotyping of rAAV Genomes with Capsids Derived from Four Additional Bocavirus Serotypes (A) BoV helper plasmid (pCMVNS*Cap1) for chimeric rAAV/HBoV1 production and acceptor plasmid (pCMVNS*CapΔ) derived thereof for cloning of the different BoV cap ORFs. Each cap sequence was ordered as two gene blocks, assembled to a full-length cap ORF (cap x , where x = HBoV2–4 or GBoV) and subsequently cloned into the acceptor plasmid using a Golden Gate reaction. BocaSR, BoV-transcribed small non-coding RNA. Numbers in brackets refer to the construct labels in Figure 1 A. (B) Production and iodixanol purification of chimeric HBoV1-4 and GBoV vectors encoding Gluc. The amount of genome copies per milliliter was determined with TaqMan RT-PCR. Shown are averages (±SEM) of four independent productions. (C) Western blot analysis of the indicated iodixanol-purified BoV stocks. Detected are the three BoV capsid proteins VP1, VP2, and VP3. NEG, iodixanol gradient from untransfected cells. (D) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 2 × 10 4 . Gluc activity in the medium was measured at 4 and 9 days post-transduction as arbitrary light units (ALU). Data are the mean Gluc expression (±SEM, n = 3). Numbers above the columns depict fold increases in expression. (E) Flow cytometry analysis of pHAEs transduced with scAAV-YFP/HBoV1 or scAAV-YFP/HBoV4. scAAV-Gluc/HBoV1 was used as a control (both at an MOI of 5 × 10 4 , n = 4 independent transwells). Cells were co-stained for YFP and a cell type-specific marker: β-Tubulin IV (ciliated cells), MUC5AC (goblet cells), CC10 (club cells), or KRT5 (basal cells). Percentages of double-positive cells are shown in the upper right quarter. (F) Transduction of primary lung organoids with the indicated scAAV-Gluc/BoV variants. Several organoids per donor were either mechanically broken (br) and incubated with a total of 5 × 10 9 viral genomes, or they were individually microinjected (in) with 5 × 10 8 –1 × 10 9 viral particles. Total Gluc activity in the medium was measured 3–12 days post-transduction and plotted on the y axis as ALU. Numbers above the columns depict fold increases in expression. (G) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 1 × 10 4 in the presence (+) or absence (−) of IVIg. Gluc activity in the medium was measured 5 days post-transduction as ALU. Shown is the mean Gluc activity (+SEM, n = 4, except for HB1 [–] n = 3). For pHAEs transduction, LLnL and Doxorubicin were added at concentrations of 40 and 5 μM, respectively. Transductions of primary lung organoids were performed in the presence of 1 μM Doxorubicin. For statistical analysis, one-way ANOVA was used. Significance at p
Figure Legend Snippet: Pseudotyping of rAAV Genomes with Capsids Derived from Four Additional Bocavirus Serotypes (A) BoV helper plasmid (pCMVNS*Cap1) for chimeric rAAV/HBoV1 production and acceptor plasmid (pCMVNS*CapΔ) derived thereof for cloning of the different BoV cap ORFs. Each cap sequence was ordered as two gene blocks, assembled to a full-length cap ORF (cap x , where x = HBoV2–4 or GBoV) and subsequently cloned into the acceptor plasmid using a Golden Gate reaction. BocaSR, BoV-transcribed small non-coding RNA. Numbers in brackets refer to the construct labels in Figure 1 A. (B) Production and iodixanol purification of chimeric HBoV1-4 and GBoV vectors encoding Gluc. The amount of genome copies per milliliter was determined with TaqMan RT-PCR. Shown are averages (±SEM) of four independent productions. (C) Western blot analysis of the indicated iodixanol-purified BoV stocks. Detected are the three BoV capsid proteins VP1, VP2, and VP3. NEG, iodixanol gradient from untransfected cells. (D) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 2 × 10 4 . Gluc activity in the medium was measured at 4 and 9 days post-transduction as arbitrary light units (ALU). Data are the mean Gluc expression (±SEM, n = 3). Numbers above the columns depict fold increases in expression. (E) Flow cytometry analysis of pHAEs transduced with scAAV-YFP/HBoV1 or scAAV-YFP/HBoV4. scAAV-Gluc/HBoV1 was used as a control (both at an MOI of 5 × 10 4 , n = 4 independent transwells). Cells were co-stained for YFP and a cell type-specific marker: β-Tubulin IV (ciliated cells), MUC5AC (goblet cells), CC10 (club cells), or KRT5 (basal cells). Percentages of double-positive cells are shown in the upper right quarter. (F) Transduction of primary lung organoids with the indicated scAAV-Gluc/BoV variants. Several organoids per donor were either mechanically broken (br) and incubated with a total of 5 × 10 9 viral genomes, or they were individually microinjected (in) with 5 × 10 8 –1 × 10 9 viral particles. Total Gluc activity in the medium was measured 3–12 days post-transduction and plotted on the y axis as ALU. Numbers above the columns depict fold increases in expression. (G) Transduction of pHAEs with the indicated scAAV-Gluc/BoV variants at an MOI of 1 × 10 4 in the presence (+) or absence (−) of IVIg. Gluc activity in the medium was measured 5 days post-transduction as ALU. Shown is the mean Gluc activity (+SEM, n = 4, except for HB1 [–] n = 3). For pHAEs transduction, LLnL and Doxorubicin were added at concentrations of 40 and 5 μM, respectively. Transductions of primary lung organoids were performed in the presence of 1 μM Doxorubicin. For statistical analysis, one-way ANOVA was used. Significance at p

Techniques Used: Derivative Assay, Plasmid Preparation, Clone Assay, Sequencing, Construct, Purification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transduction, Activity Assay, Expressing, Flow Cytometry, Cytometry, Staining, Marker, Incubation

Transduction of Primary Human Hepatocytes and T Cells with rAAV/BoV Vectors (A) YFP expression 5 days after transduction of primary human hepatocytes (pHeps) with scAAV-YFP/BoV vectors at the indicated MOIs (n = 3 donors). Expression first became detectable at day 2 (not shown) and then increased over time. Cells were fixed and stained with an FITC-coupled anti-GFP antibody, and nuclei were stained with Hoechst. Scale bar, 50 μm. (B) Gluc activity at 3 and 6 days post-transduction of pHeps with scAAV-Gluc/BoV at an MOI of 1 × 10 4 . Data represent the mean and range of two independent measurements (n = 2 donors). (C) Transduction of primary human T cells with scAAV-Gluc/BoV (MOI = 1 × 10 4 ). Gluc activity in the medium was measured 3–12 days post-transduction. Plotted are means ±SEM of three independent experiments (n = 3 donors). For all transductions, Doxorubicin was added to a final concentration of 1 μM/well. Dashed lines indicate the assay background.
Figure Legend Snippet: Transduction of Primary Human Hepatocytes and T Cells with rAAV/BoV Vectors (A) YFP expression 5 days after transduction of primary human hepatocytes (pHeps) with scAAV-YFP/BoV vectors at the indicated MOIs (n = 3 donors). Expression first became detectable at day 2 (not shown) and then increased over time. Cells were fixed and stained with an FITC-coupled anti-GFP antibody, and nuclei were stained with Hoechst. Scale bar, 50 μm. (B) Gluc activity at 3 and 6 days post-transduction of pHeps with scAAV-Gluc/BoV at an MOI of 1 × 10 4 . Data represent the mean and range of two independent measurements (n = 2 donors). (C) Transduction of primary human T cells with scAAV-Gluc/BoV (MOI = 1 × 10 4 ). Gluc activity in the medium was measured 3–12 days post-transduction. Plotted are means ±SEM of three independent experiments (n = 3 donors). For all transductions, Doxorubicin was added to a final concentration of 1 μM/well. Dashed lines indicate the assay background.

Techniques Used: Transduction, Expressing, Staining, Activity Assay, Concentration Assay

10) Product Images from "MicroRNA-452 promotes stem-like cells of hepatocellular carcinoma by inhibiting Sox7 involving Wnt/β-catenin signaling pathway"

Article Title: MicroRNA-452 promotes stem-like cells of hepatocellular carcinoma by inhibiting Sox7 involving Wnt/β-catenin signaling pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.8584

Combination of doxorubicin and ATRA chemotherapy efficiently suppressed metastasis of miR-452 overexpressed HCC Apoptosis of ( A ) LM3/miR-452 and ( B ) HuH7/miR-452 cells receiving the indicated treatments was assessed by Annexin V-APC staining. ( C ) The cell viability was detected after treatment in different groups by CCK8. ( D ) The treatment of in vivo metastasis model with doxorubicin and/or ATRA were performed using LM3/miR-452 cells ( n = 5). ( E ) Further, the average number and total weight of intrahepatic metastatic nodules were showed. ( F ) The percentage of CD44+ and CD133+ subpopulation CSCs were detected derived from those metastatic nodules by FACS.
Figure Legend Snippet: Combination of doxorubicin and ATRA chemotherapy efficiently suppressed metastasis of miR-452 overexpressed HCC Apoptosis of ( A ) LM3/miR-452 and ( B ) HuH7/miR-452 cells receiving the indicated treatments was assessed by Annexin V-APC staining. ( C ) The cell viability was detected after treatment in different groups by CCK8. ( D ) The treatment of in vivo metastasis model with doxorubicin and/or ATRA were performed using LM3/miR-452 cells ( n = 5). ( E ) Further, the average number and total weight of intrahepatic metastatic nodules were showed. ( F ) The percentage of CD44+ and CD133+ subpopulation CSCs were detected derived from those metastatic nodules by FACS.

Techniques Used: Staining, In Vivo, Derivative Assay, FACS

Up-regulation of miR-452 in HCC ( A ) The percentage of CD44+ and CD133+ cells markedly increased upon miR-452 up-regulation both in LM3 and Huh7. ( B ) MiR-452 overexpressed HCC cells showed higher chemoresistance to doxorubicin and sorafenib. ( C ) The self-renewal capability of LM3 and Huh7 was significantly enhanced after miR-452 expression increased in tumor sphere assay. ( D ) miR-452 efficiently promoted HCC invasion in vitro . ( E ) The capability of tumorigenicity significantly enhanced both in the primary and secondary xenograft model of LM3/miR-452 cells in comparison with LM3/NC cells. ( F ) In vivo metastasis assay, miR-452 distinctly aggravated the metastasis of LM3 cells ( n = 5).
Figure Legend Snippet: Up-regulation of miR-452 in HCC ( A ) The percentage of CD44+ and CD133+ cells markedly increased upon miR-452 up-regulation both in LM3 and Huh7. ( B ) MiR-452 overexpressed HCC cells showed higher chemoresistance to doxorubicin and sorafenib. ( C ) The self-renewal capability of LM3 and Huh7 was significantly enhanced after miR-452 expression increased in tumor sphere assay. ( D ) miR-452 efficiently promoted HCC invasion in vitro . ( E ) The capability of tumorigenicity significantly enhanced both in the primary and secondary xenograft model of LM3/miR-452 cells in comparison with LM3/NC cells. ( F ) In vivo metastasis assay, miR-452 distinctly aggravated the metastasis of LM3 cells ( n = 5).

Techniques Used: Expressing, In Vitro, In Vivo

MiR-452 indicated poor survival in HCC ( A ) Hepatospheres were obtained from HepG2 cells after 20 serial passages in serum-free medium combined with doxorubicin and sorafenib. Hepatospheres with ( B ) higher capability of tumorigenicity, ( C ) higher expression of stemness-related genes, and ( D ) markedly overexpression of miR-452 compared with the differentiated clones. ( E ) Analysis of miR-452 expression in adjacent normal liver tissues and HCC specimens in accordance with different TNM stages both in the training and validation cohort. ( F ) Kaplan Meier analysis of HCC patients with high- versus low-expression of miR-452 both in training cohort ( P
Figure Legend Snippet: MiR-452 indicated poor survival in HCC ( A ) Hepatospheres were obtained from HepG2 cells after 20 serial passages in serum-free medium combined with doxorubicin and sorafenib. Hepatospheres with ( B ) higher capability of tumorigenicity, ( C ) higher expression of stemness-related genes, and ( D ) markedly overexpression of miR-452 compared with the differentiated clones. ( E ) Analysis of miR-452 expression in adjacent normal liver tissues and HCC specimens in accordance with different TNM stages both in the training and validation cohort. ( F ) Kaplan Meier analysis of HCC patients with high- versus low-expression of miR-452 both in training cohort ( P

Techniques Used: Expressing, Over Expression, Clone Assay

11) Product Images from "Autophagic homeostasis is required for the pluripotency of cancer stem cells"

Article Title: Autophagic homeostasis is required for the pluripotency of cancer stem cells

Journal: Autophagy

doi: 10.1080/15548627.2016.1260808

Senescence induction via doxorubicin decreases pluripotency, increases differentiation, and inhibits autophagy in NT2/D1 cells. (A) NT2/D1 cells treated with 10 nM of doxorubicin were subjected to WB analysis for CDKN1A (B) and cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B . (C) GLB1 staining was performed to confirm the presence of senescent cells following 3 nM doxorubicin treatment. (D) Cells treated with 3 nM of doxorubicin were stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h post-treatment. (E) Photographs were taken of cells treated with 3 nM and 5 nM of doxorubicin to show changes in cell morphology compare with the untreated control. (F) Cells treated with 10 nM of doxorubicin were subjected to WB analysis for pluripotency factors (POU5F1, NANOG, SOX2) and differentiation markers (TUBB3). (G) Cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for pluripotency factors ( POU5F1, NANOG, SOX2 ) and differentiation markers ( TUBB3, CSN2, GATA6, CDX2 ). (H) Cells treated with 10 nM of doxorubicin were subjected to WB analysis for autophagy markers (ATG5, ATG7, SQSTM1, LC3A-II, LC3B-II and p-MTOR). (I) Cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for autophagy markers ( ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 ). (J) NT2/D1 cells treated with doxorubicin (10 nM), chloroquine (CQ) (18 µM) or in combination were subjected to WB analysis for protein levels of SQSTM1, LC3A-II and LC3B-II. Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. Scale bar: 100 µm.
Figure Legend Snippet: Senescence induction via doxorubicin decreases pluripotency, increases differentiation, and inhibits autophagy in NT2/D1 cells. (A) NT2/D1 cells treated with 10 nM of doxorubicin were subjected to WB analysis for CDKN1A (B) and cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for CDKN1A, CDKN2A and CDKN1B . (C) GLB1 staining was performed to confirm the presence of senescent cells following 3 nM doxorubicin treatment. (D) Cells treated with 3 nM of doxorubicin were stained with trypan blue and counted to determine the number of viable cells after 24 and 48 h post-treatment. (E) Photographs were taken of cells treated with 3 nM and 5 nM of doxorubicin to show changes in cell morphology compare with the untreated control. (F) Cells treated with 10 nM of doxorubicin were subjected to WB analysis for pluripotency factors (POU5F1, NANOG, SOX2) and differentiation markers (TUBB3). (G) Cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for pluripotency factors ( POU5F1, NANOG, SOX2 ) and differentiation markers ( TUBB3, CSN2, GATA6, CDX2 ). (H) Cells treated with 10 nM of doxorubicin were subjected to WB analysis for autophagy markers (ATG5, ATG7, SQSTM1, LC3A-II, LC3B-II and p-MTOR). (I) Cells treated with 3 nM of doxorubicin were subjected to qRT-PCR analysis for autophagy markers ( ATG5, ATG7, ATG12, BECN1, ULK1 and SQSTM1 ). (J) NT2/D1 cells treated with doxorubicin (10 nM), chloroquine (CQ) (18 µM) or in combination were subjected to WB analysis for protein levels of SQSTM1, LC3A-II and LC3B-II. Statistical analysis was performed with the 2-tailed, Student t test with 95% confidence interval; * P values ≤ 0.05 obtained by comparing the respective data with the untreated or scrambled control. Scale bar: 100 µm.

Techniques Used: Western Blot, Quantitative RT-PCR, Staining

12) Product Images from "Fuzzy Tandem Repeats Containing p53 Response Elements May Define Species-Specific p53 Target Genes"

Article Title: Fuzzy Tandem Repeats Containing p53 Response Elements May Define Species-Specific p53 Target Genes

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002731

Transactivation of murine Ncoa1 by p53 relies on a cluster of p53 half-sites. (A) Sequence of the clustered p53 REs upstream of the Ncoa1 gene, represented as in Figure 1D . Numbers are relative to the TSS. The parentheses indicate a putative half-site with a rare variant of the core CWWG motif: evidence that p53 may bind a half-site with a CWWA core was reported [42] , but only 2% of the sequences bound by p53 contain a p53RE with a CWWA core in its second half-site [19] . (B) MEFs were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. Results, from 7 experiments, were plotted as in Figure 1A . (C) A 2.5 kb-long fragment containing sequences upstream of the Ncoa1 TSS (from −3.5 kb to −1 kb) was cloned before a luciferase reporter gene, and transfected in p53 −/− MEFs alone ( IV ), or with an expression vector for WT p53 ( IV +p53WT), or mutant p53 ( IV +p53R270H), then luciferase activities were measured. Luciferase activities were determined with the same sequences after deletion of the clustered putative p53 REs (sequences from −3.1 kb to −1 kb; plasmid V ). Results, from 3 independent experiments, were normalized to control renilla luciferase, then plotted as in Figure 1E . (D) ChIP assay was performed in doxorubicin-treated p53 −/− and wild-type MEFs, with an antibody against p53 or rabbit IgG as a control. Immunoprecipitates, from 3 independent experiments, were quantified and plotted as above. (E) Fuzzy tandem repeats within the Ncoa1 cluster. Integrated mreps results are represented as in Figure 1G . (F) The cluster of p53 REs at the Ncoa1 locus is poorly conserved in evolution. Candidate p53 REs were searched for at the murine and human Ncoa1 loci and plotted as before. (G) Human fibroblasts were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. After stress, p21 mRNAs levels increased ( Figure 1H ), but not Ncoa1 mRNA levels. Results, from 3 independent experiments, were normalized and plotted as before.
Figure Legend Snippet: Transactivation of murine Ncoa1 by p53 relies on a cluster of p53 half-sites. (A) Sequence of the clustered p53 REs upstream of the Ncoa1 gene, represented as in Figure 1D . Numbers are relative to the TSS. The parentheses indicate a putative half-site with a rare variant of the core CWWG motif: evidence that p53 may bind a half-site with a CWWA core was reported [42] , but only 2% of the sequences bound by p53 contain a p53RE with a CWWA core in its second half-site [19] . (B) MEFs were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. Results, from 7 experiments, were plotted as in Figure 1A . (C) A 2.5 kb-long fragment containing sequences upstream of the Ncoa1 TSS (from −3.5 kb to −1 kb) was cloned before a luciferase reporter gene, and transfected in p53 −/− MEFs alone ( IV ), or with an expression vector for WT p53 ( IV +p53WT), or mutant p53 ( IV +p53R270H), then luciferase activities were measured. Luciferase activities were determined with the same sequences after deletion of the clustered putative p53 REs (sequences from −3.1 kb to −1 kb; plasmid V ). Results, from 3 independent experiments, were normalized to control renilla luciferase, then plotted as in Figure 1E . (D) ChIP assay was performed in doxorubicin-treated p53 −/− and wild-type MEFs, with an antibody against p53 or rabbit IgG as a control. Immunoprecipitates, from 3 independent experiments, were quantified and plotted as above. (E) Fuzzy tandem repeats within the Ncoa1 cluster. Integrated mreps results are represented as in Figure 1G . (F) The cluster of p53 REs at the Ncoa1 locus is poorly conserved in evolution. Candidate p53 REs were searched for at the murine and human Ncoa1 loci and plotted as before. (G) Human fibroblasts were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. After stress, p21 mRNAs levels increased ( Figure 1H ), but not Ncoa1 mRNA levels. Results, from 3 independent experiments, were normalized and plotted as before.

Techniques Used: Sequencing, Variant Assay, RNA Extraction, Real-time Polymerase Chain Reaction, Clone Assay, Luciferase, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation

Murine Rbl2/ p130 is a p53 target gene. (A) WT and p53 −/− MEFs were left untreated (Unt) or treated with doxorubicin (Dox) before RNA extraction and real-time PCR quantification, in 8 independent experiments. Data were normalized to control mRNA levels, then a value of 1 was assigned to mRNA amounts in unstressed WT cells. (B) MEFs were treated as in (A), then protein extracts were immunoblotted with antibodies to p130, p53 and Gapdh. (C) MEFs were left untreated (Unt) or treated with Nutlin (Nut) before RNA quantification, in 4 independent experiments. (D) Putative p53 REs, identified using Consite and a positional frequency matrix ( Methods ), were plotted along the Rbl2 /p130 locus as lollipops, with greytones according to scores ( Table S1 ). Numbers are relative to the transcription start site (TSS). Black box: exon 1. Below, the cluster sequence is shown, with p53 putative binding half-sites in bold (putative binding sites have 0–3 mismatches with the consensus; matches in capital letters and mismatches in lowercase; perfect half-sites are boxed). (E) The 6 kb upstream the TSS were cloned before a luciferase reporter gene, and the plasmid was transfected in p53 −/− MEFs alone ( I ), or with an expression vector for WT p53 ( I +p53WT) or mutant p53 ( I +p53R270H) to measure luciferase. Likewise, luciferase was measured with plasmids containing 2.5 kb of sequences downstream the TSS with ( II ) or without ( III ) the clustered p53 REs. Results, from 3 independent experiments, were normalized to control renilla luciferase, then a value of 1 was assigned to luciferase in cells transfected with reporter plasmids alone. (F) ChIP assay was performed in doxorubicin-treated MEFs, with an antibody against p53, or rabbit IgG as a control. Immunoprecipitates were quantified using real-time PCR and normalized to input DNA on an irrelevant region, in 3 independent experiments. (G) Integrated results of the cluster sequence analysis with mreps . Numbers are relative to the TSS. (H) Human lung fibroblasts were treated and mRNAs were quantified as in (A), in 3 independent experiments. Similar results were obtained with foreskin fibroblasts. (I) Putative p53 response elements were searched for and plotted along the human Rbl2 /p130 locus as in (D). The region homologous to the murine clustered p53 REs is below, with putative half-sites as before (those with a single mismatch are underlined). The parentheses indicate a nonamer that might be a putative half-site with a deletion within the core CWWG motif (the minus sign indicates the deletion), a situation observed in about 5% of p53 binding half-sites [41] .
Figure Legend Snippet: Murine Rbl2/ p130 is a p53 target gene. (A) WT and p53 −/− MEFs were left untreated (Unt) or treated with doxorubicin (Dox) before RNA extraction and real-time PCR quantification, in 8 independent experiments. Data were normalized to control mRNA levels, then a value of 1 was assigned to mRNA amounts in unstressed WT cells. (B) MEFs were treated as in (A), then protein extracts were immunoblotted with antibodies to p130, p53 and Gapdh. (C) MEFs were left untreated (Unt) or treated with Nutlin (Nut) before RNA quantification, in 4 independent experiments. (D) Putative p53 REs, identified using Consite and a positional frequency matrix ( Methods ), were plotted along the Rbl2 /p130 locus as lollipops, with greytones according to scores ( Table S1 ). Numbers are relative to the transcription start site (TSS). Black box: exon 1. Below, the cluster sequence is shown, with p53 putative binding half-sites in bold (putative binding sites have 0–3 mismatches with the consensus; matches in capital letters and mismatches in lowercase; perfect half-sites are boxed). (E) The 6 kb upstream the TSS were cloned before a luciferase reporter gene, and the plasmid was transfected in p53 −/− MEFs alone ( I ), or with an expression vector for WT p53 ( I +p53WT) or mutant p53 ( I +p53R270H) to measure luciferase. Likewise, luciferase was measured with plasmids containing 2.5 kb of sequences downstream the TSS with ( II ) or without ( III ) the clustered p53 REs. Results, from 3 independent experiments, were normalized to control renilla luciferase, then a value of 1 was assigned to luciferase in cells transfected with reporter plasmids alone. (F) ChIP assay was performed in doxorubicin-treated MEFs, with an antibody against p53, or rabbit IgG as a control. Immunoprecipitates were quantified using real-time PCR and normalized to input DNA on an irrelevant region, in 3 independent experiments. (G) Integrated results of the cluster sequence analysis with mreps . Numbers are relative to the TSS. (H) Human lung fibroblasts were treated and mRNAs were quantified as in (A), in 3 independent experiments. Similar results were obtained with foreskin fibroblasts. (I) Putative p53 response elements were searched for and plotted along the human Rbl2 /p130 locus as in (D). The region homologous to the murine clustered p53 REs is below, with putative half-sites as before (those with a single mismatch are underlined). The parentheses indicate a nonamer that might be a putative half-site with a deletion within the core CWWG motif (the minus sign indicates the deletion), a situation observed in about 5% of p53 binding half-sites [41] .

Techniques Used: RNA Extraction, Real-time Polymerase Chain Reaction, Sequencing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Expressing, Mutagenesis, Chromatin Immunoprecipitation

p53 transactivates murine Klhl26 via a cluster of p53 half-sites. (A) Sequence of the cluster of p53 half-sites in Klhl26 intron 1, represented as in Figure 1D . Numbers are relative to the TSS. (B) WT and p53 −/− MEFs were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. Results from 3 independent experiments. (C) A 1 kb-long fragment from Klhl26 intron 1 (from +0.1 kb to +1.1 kb) was cloned before a luciferase reporter gene, to test for p53-dependent reporter activity. The reporter plasmid was transfected in p53 −/− MEFs alone ( VI ), together with an expression vector for p53 WT ( VI +p53WT) or for mutant p53 ( VI +p53R270H) and luciferase activities were measured. Similarly, luciferase activities were determined with the same sequences after deletion of clustered putative p53 half-sites (sequences from +0.39 to +0.64 kb were deleted; plasmid VII ). Results, from 3 independent experiments, were normalized to control renilla luciferase, then plotted as in Figure 1E . (D) ChIP assay was performed in doxorubicin-treated p53 −/− and wild-type MEFs, with an antibody against p53 or rabbit IgG as a control. Immunoprecipitates, from 3 independent experiments, were quantified and plotted as before. (E) Integrated results of the mreps DNA sequence analysis, represented as in Figure 1G . (F) The cluster of p53 REs at the Klhl26 locus is poorly conserved in evolution. Candidate p53 REs were searched for at the murine and human Klhl26 loci and plotted as before. (G) Klhl26 is not induced in response to stress in human fibroblasts. Human fibroblasts were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and quantification. After stress, p21 mRNAs levels increased ( Figure 1H ), but not Klhl26 mRNA levels. Results, from 4 independent experiments, were normalized and plotted as before.
Figure Legend Snippet: p53 transactivates murine Klhl26 via a cluster of p53 half-sites. (A) Sequence of the cluster of p53 half-sites in Klhl26 intron 1, represented as in Figure 1D . Numbers are relative to the TSS. (B) WT and p53 −/− MEFs were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and real-time PCR quantification. Results from 3 independent experiments. (C) A 1 kb-long fragment from Klhl26 intron 1 (from +0.1 kb to +1.1 kb) was cloned before a luciferase reporter gene, to test for p53-dependent reporter activity. The reporter plasmid was transfected in p53 −/− MEFs alone ( VI ), together with an expression vector for p53 WT ( VI +p53WT) or for mutant p53 ( VI +p53R270H) and luciferase activities were measured. Similarly, luciferase activities were determined with the same sequences after deletion of clustered putative p53 half-sites (sequences from +0.39 to +0.64 kb were deleted; plasmid VII ). Results, from 3 independent experiments, were normalized to control renilla luciferase, then plotted as in Figure 1E . (D) ChIP assay was performed in doxorubicin-treated p53 −/− and wild-type MEFs, with an antibody against p53 or rabbit IgG as a control. Immunoprecipitates, from 3 independent experiments, were quantified and plotted as before. (E) Integrated results of the mreps DNA sequence analysis, represented as in Figure 1G . (F) The cluster of p53 REs at the Klhl26 locus is poorly conserved in evolution. Candidate p53 REs were searched for at the murine and human Klhl26 loci and plotted as before. (G) Klhl26 is not induced in response to stress in human fibroblasts. Human fibroblasts were left untreated (Unt) or treated with doxorubicin (Dox) for 24 h, before RNA extraction and quantification. After stress, p21 mRNAs levels increased ( Figure 1H ), but not Klhl26 mRNA levels. Results, from 4 independent experiments, were normalized and plotted as before.

Techniques Used: Sequencing, RNA Extraction, Real-time Polymerase Chain Reaction, Clone Assay, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Expressing, Mutagenesis, Chromatin Immunoprecipitation

A partial divergence in the regulation of Rbl2 /p130, Ncoa1 , and Klhl26 among rodents. (A) Homologous regions of the Rbl2 , Ncoa1 and Klhl26 loci from mouse and rat were analyzed with Consite, and results were plotted along the map as before. At each rat locus, 0–2 putative p53 REs were found to map at the same position as a cluster of p53 REs at the homologous murine locus region. Their sequence is indicated below the map. (B) Primary REFs were left untreated (Unt) or treated for 24 h with doxorubicin (Dox) at 0.5 µg/ml, or Nutlin at 10 µM, before RNA extraction and real-time PCR quantification. Results were normalized to control mRNA levels, then the mean amount of mRNAs in unstressed cells was assigned a value of 1. Data are from 3 independent experiments.
Figure Legend Snippet: A partial divergence in the regulation of Rbl2 /p130, Ncoa1 , and Klhl26 among rodents. (A) Homologous regions of the Rbl2 , Ncoa1 and Klhl26 loci from mouse and rat were analyzed with Consite, and results were plotted along the map as before. At each rat locus, 0–2 putative p53 REs were found to map at the same position as a cluster of p53 REs at the homologous murine locus region. Their sequence is indicated below the map. (B) Primary REFs were left untreated (Unt) or treated for 24 h with doxorubicin (Dox) at 0.5 µg/ml, or Nutlin at 10 µM, before RNA extraction and real-time PCR quantification. Results were normalized to control mRNA levels, then the mean amount of mRNAs in unstressed cells was assigned a value of 1. Data are from 3 independent experiments.

Techniques Used: Sequencing, RNA Extraction, Real-time Polymerase Chain Reaction

13) Product Images from "A Kinome-Wide RNAi Screen in Drosophila Glia Reveals That the RIO Kinases Mediate Cell Proliferation and Survival through TORC2-Akt Signaling in Glioblastoma"

Article Title: A Kinome-Wide RNAi Screen in Drosophila Glia Reveals That the RIO Kinases Mediate Cell Proliferation and Survival through TORC2-Akt Signaling in Glioblastoma

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003253

Loss of RIOK1 or RIOK2 function chemosensitizes GBM cells and reduces TORC2-Akt signaling. (A) Knockdown of RIOK1 or RIOK2 sensitizes GBM cells to apoptosis in response to treatment with doxorubicin (doxo) and temozolomide (tmz), as evidenced by blots for active caspase-3 and PARP cleavage (A). All samples blotted for RIOK1 and RIOK2 to confirm changes in RIOK1 levels with RIOK2 knockdown, evident in p53-wild-type GBM cell lines. The RIOKs also decline with doxorubicin treatment. GBM301 cells were treated for 24 hrs with 1 µg/mL doxorubicin beginning 96 hrs post infection with viral vectors. U87MG, A172, and LNZ308 cells were treated for 24 hrs with 1 µg/mL doxorubicin and 100 µM temozolomide beginning 72 hrs post transfection with siRNAs. (B) FACS-based quantification of chemosensitivity. 96 hours post shRNA infection, U87MG samples were split in half and treated for 12 hours with either DMSO (light blue) or 1 µg/mL doxorubicin and 100 µM temozolomide (red). Live cells were collected and stained for 7AAD and Annexin-V. Data is represented as the percentage of Annexin V-positive 7AAD-negative cells in each sample, averaged over 2 experiments. P-values refer to student's two-tailed t-test used to compare doxorubicin and temozolomide-treated control to RIOK-shRNA cells. Validation of knockdown shown. FACS plots and raw data shown in Figure S15 . (C) GBM301 cells treated with 25 µM ZVAD for 48 hrs beginning 3 days post-infection with viral vectors. Reduced phosphorylation of Akt on the TORC2 target site, Serine-473, is visible relative to total Akt protein. Reduced phosphorylation of several Akt targets, such as the FOXO3 transcription factor, is clear when phospho-epitope signal is compared to total protein controls. PARP cleavage is a read-out for apoptosis; PARP cleavage fragment in RIOK2 knockdown cells indicates residual caspase activity, due to the strong effect of RIOK2 loss. p53 upregulation was evident in GBM301 cells in the absence of residual caspase activity.
Figure Legend Snippet: Loss of RIOK1 or RIOK2 function chemosensitizes GBM cells and reduces TORC2-Akt signaling. (A) Knockdown of RIOK1 or RIOK2 sensitizes GBM cells to apoptosis in response to treatment with doxorubicin (doxo) and temozolomide (tmz), as evidenced by blots for active caspase-3 and PARP cleavage (A). All samples blotted for RIOK1 and RIOK2 to confirm changes in RIOK1 levels with RIOK2 knockdown, evident in p53-wild-type GBM cell lines. The RIOKs also decline with doxorubicin treatment. GBM301 cells were treated for 24 hrs with 1 µg/mL doxorubicin beginning 96 hrs post infection with viral vectors. U87MG, A172, and LNZ308 cells were treated for 24 hrs with 1 µg/mL doxorubicin and 100 µM temozolomide beginning 72 hrs post transfection with siRNAs. (B) FACS-based quantification of chemosensitivity. 96 hours post shRNA infection, U87MG samples were split in half and treated for 12 hours with either DMSO (light blue) or 1 µg/mL doxorubicin and 100 µM temozolomide (red). Live cells were collected and stained for 7AAD and Annexin-V. Data is represented as the percentage of Annexin V-positive 7AAD-negative cells in each sample, averaged over 2 experiments. P-values refer to student's two-tailed t-test used to compare doxorubicin and temozolomide-treated control to RIOK-shRNA cells. Validation of knockdown shown. FACS plots and raw data shown in Figure S15 . (C) GBM301 cells treated with 25 µM ZVAD for 48 hrs beginning 3 days post-infection with viral vectors. Reduced phosphorylation of Akt on the TORC2 target site, Serine-473, is visible relative to total Akt protein. Reduced phosphorylation of several Akt targets, such as the FOXO3 transcription factor, is clear when phospho-epitope signal is compared to total protein controls. PARP cleavage is a read-out for apoptosis; PARP cleavage fragment in RIOK2 knockdown cells indicates residual caspase activity, due to the strong effect of RIOK2 loss. p53 upregulation was evident in GBM301 cells in the absence of residual caspase activity.

Techniques Used: Infection, Transfection, FACS, shRNA, Staining, Two Tailed Test, Activity Assay

14) Product Images from "GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy"

Article Title: GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

Journal: Autophagy

doi: 10.1080/15548627.2016.1239676

Cisplatin induces GFRA1 expression in osteosarcoma cells. (A to C) Immunoblot analysis of osteosarcoma cell lysates with antibodies specific for GFRA1 and ACTB/β-actin. (A) MG-63 and U-2 OS cells were treated with doxorubicin (5 μM),
Figure Legend Snippet: Cisplatin induces GFRA1 expression in osteosarcoma cells. (A to C) Immunoblot analysis of osteosarcoma cell lysates with antibodies specific for GFRA1 and ACTB/β-actin. (A) MG-63 and U-2 OS cells were treated with doxorubicin (5 μM),

Techniques Used: Expressing

15) Product Images from "Senoptosis: non-lethal DNA cleavage as a route to deep senescence"

Article Title: Senoptosis: non-lethal DNA cleavage as a route to deep senescence

Journal: Oncotarget

doi: 10.18632/oncotarget.15693

DNA content, growth and cell death analysis of MRC5 cells for different γ-irradiation regimes A . Western blot analysis of MRC5 whole cell extracts isolated from irradiated (10Gy) MRC5 cells harvested at day 0, 0.5, 1, 3, 5, and 7. Immunoblot was probed for proteins activated after DNA damage. B . Frequency histogram of DNA content (in cytometric measurements equivalent to DNA stain fluorescence) presenting the proportion of the cell population within the sub-G1, G1, and G2+M phases. Lower panel: corresponding SSC-A vs . FCS-A (A- area) scatter plots with gated viable, single cells. C . Time series of population doublings for different γ-radiation regimes and treatments with doxorubicin (1μM for 6 hours), etoposide (50μM for 8 hours), or staurosporine (1μM for 4 hours) (mean ± SEM ( n ≥ 3), cell counts > 100 cells) D . Time series for the sub-G1 percentages in MRC5 fibroblasts after different γ-irradiation regimes or treatment with doxorubicin, etoposide, and staurosporine (mean ± SEM ( n = 3)). DOX- doxorubicin, ETO- etoposide, STS- staurosporine. E . Bar graphs representing percentage of Annexin V/PI cell positive cells over seven days after irradiation or 1 day after staurosporine treatment (STS). Live cells (negative for both Annexin V (AV) and propidium iodide (PI), early apoptotic cells (positive for Annexin V and negative for PI), late apoptotic/necrotic cells (positive for both Annexin V and PI) and dead cells (negative for Annexin V and positive for PI), (mean ± SEM ( n = 3)).
Figure Legend Snippet: DNA content, growth and cell death analysis of MRC5 cells for different γ-irradiation regimes A . Western blot analysis of MRC5 whole cell extracts isolated from irradiated (10Gy) MRC5 cells harvested at day 0, 0.5, 1, 3, 5, and 7. Immunoblot was probed for proteins activated after DNA damage. B . Frequency histogram of DNA content (in cytometric measurements equivalent to DNA stain fluorescence) presenting the proportion of the cell population within the sub-G1, G1, and G2+M phases. Lower panel: corresponding SSC-A vs . FCS-A (A- area) scatter plots with gated viable, single cells. C . Time series of population doublings for different γ-radiation regimes and treatments with doxorubicin (1μM for 6 hours), etoposide (50μM for 8 hours), or staurosporine (1μM for 4 hours) (mean ± SEM ( n ≥ 3), cell counts > 100 cells) D . Time series for the sub-G1 percentages in MRC5 fibroblasts after different γ-irradiation regimes or treatment with doxorubicin, etoposide, and staurosporine (mean ± SEM ( n = 3)). DOX- doxorubicin, ETO- etoposide, STS- staurosporine. E . Bar graphs representing percentage of Annexin V/PI cell positive cells over seven days after irradiation or 1 day after staurosporine treatment (STS). Live cells (negative for both Annexin V (AV) and propidium iodide (PI), early apoptotic cells (positive for Annexin V and negative for PI), late apoptotic/necrotic cells (positive for both Annexin V and PI) and dead cells (negative for Annexin V and positive for PI), (mean ± SEM ( n = 3)).

Techniques Used: Irradiation, Western Blot, Isolation, Staining, Fluorescence

16) Product Images from "Phosphorylation-Dependent Degradation of p300 by Doxorubicin-Activated p38 Mitogen-Activated Protein Kinase in Cardiac Cells"

Article Title: Phosphorylation-Dependent Degradation of p300 by Doxorubicin-Activated p38 Mitogen-Activated Protein Kinase in Cardiac Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.7.2673-2687.2005

Suppression of doxorubicin-induced p300 degradation by inhibition of p38 MAPK. (A) Primary cardiomyocytes were treated with various concentrations of SB 202190, and p300 protein levels were determined by Western blot analysis with an anti-p300 antibody.
Figure Legend Snippet: Suppression of doxorubicin-induced p300 degradation by inhibition of p38 MAPK. (A) Primary cardiomyocytes were treated with various concentrations of SB 202190, and p300 protein levels were determined by Western blot analysis with an anti-p300 antibody.

Techniques Used: Inhibition, Western Blot

Doxorubicin induces phosphorylation of p300 in cardiomyocytes in culture, and inhibition of the proteasome activity reduces degradation of phosphorylated p300. (A) Primary cardiomyocytes were maintained in medium without phosphate and then labeled with
Figure Legend Snippet: Doxorubicin induces phosphorylation of p300 in cardiomyocytes in culture, and inhibition of the proteasome activity reduces degradation of phosphorylated p300. (A) Primary cardiomyocytes were maintained in medium without phosphate and then labeled with

Techniques Used: Inhibition, Activity Assay, Labeling

p300 degradation in cardiomyocytes treated with doxorubicin parallels apoptosis. (A) Primary neonatal cardiomyocytes plated on coverslides were maintained in normal medium (Untr.) or in medium supplemented with 1 μM doxorubicin for 24 h (Dox 24
Figure Legend Snippet: p300 degradation in cardiomyocytes treated with doxorubicin parallels apoptosis. (A) Primary neonatal cardiomyocytes plated on coverslides were maintained in normal medium (Untr.) or in medium supplemented with 1 μM doxorubicin for 24 h (Dox 24

Techniques Used:

p300 degradation and stabilization of the p53 protein after doxorubicin treatment. Primary neonatal cardiomyocytes plated on coverslides were maintained in normal medium (control) or in medium supplemented with 1 μM doxorubicin for 24 h (Dox 24
Figure Legend Snippet: p300 degradation and stabilization of the p53 protein after doxorubicin treatment. Primary neonatal cardiomyocytes plated on coverslides were maintained in normal medium (control) or in medium supplemented with 1 μM doxorubicin for 24 h (Dox 24

Techniques Used:

Loss of GATA-4 following doxorubicin exposure is not mediated by depletion of p300 protein. Primary neonatal rat cardiomyocytes were isolated and maintained in normal medium in medium supplemented with 1 μM doxorubicin (Dox), or in medium containing
Figure Legend Snippet: Loss of GATA-4 following doxorubicin exposure is not mediated by depletion of p300 protein. Primary neonatal rat cardiomyocytes were isolated and maintained in normal medium in medium supplemented with 1 μM doxorubicin (Dox), or in medium containing

Techniques Used: Isolation

Model of p38-mediated degradation of p300. On treatment with doxorubicin (Dox), neonatal cardiomyocytes up-regulate the activity of p38, which targets the N-terminal and C-terminal regions of p300 for phosphorylation. Phosphorylated p300 is then targeted
Figure Legend Snippet: Model of p38-mediated degradation of p300. On treatment with doxorubicin (Dox), neonatal cardiomyocytes up-regulate the activity of p38, which targets the N-terminal and C-terminal regions of p300 for phosphorylation. Phosphorylated p300 is then targeted

Techniques Used: Activity Assay

A serine/threonine inhibitor stabilizes the p300 protein level, and doxorubicin (Dox) treatment induces threonine phosphorylation of p300 in cardiomyocytes. (A) Primary neonatal cardiomyocytes were treated with 1 nM staurosporine or 3 μM H-7 and
Figure Legend Snippet: A serine/threonine inhibitor stabilizes the p300 protein level, and doxorubicin (Dox) treatment induces threonine phosphorylation of p300 in cardiomyocytes. (A) Primary neonatal cardiomyocytes were treated with 1 nM staurosporine or 3 μM H-7 and

Techniques Used:

Doxorubicin activates pp38 in cardiomyocytes. Primary neonatal cardiomyocytes were plated on coverslides and maintained in normal medium (Untr.) or treated with 1 μM doxorubicin for 12 or 24 h (Dox 12 h, Dox 24 h). (A and B) The cells were incubated
Figure Legend Snippet: Doxorubicin activates pp38 in cardiomyocytes. Primary neonatal cardiomyocytes were plated on coverslides and maintained in normal medium (Untr.) or treated with 1 μM doxorubicin for 12 or 24 h (Dox 12 h, Dox 24 h). (A and B) The cells were incubated

Techniques Used: Incubation

Interaction of p300 and p38 in cardiomyocytes in vivo. Primary neonatal cardiomyocytes were maintained for 12 h in normal medium or in medium supplemented with 1 μM doxorubicin (Dox). Nuclear extracts were prepared, and equal amounts of nuclear
Figure Legend Snippet: Interaction of p300 and p38 in cardiomyocytes in vivo. Primary neonatal cardiomyocytes were maintained for 12 h in normal medium or in medium supplemented with 1 μM doxorubicin (Dox). Nuclear extracts were prepared, and equal amounts of nuclear

Techniques Used: In Vivo

17) Product Images from "Real-time Imaging and Quantitative Analysis of Doxorubicin Transport in a Perfusable Microvessel Platform"

Article Title: Real-time Imaging and Quantitative Analysis of Doxorubicin Transport in a Perfusable Microvessel Platform

Journal: Integrative biology : quantitative biosciences from nano to macro

doi: 10.1039/c6ib00082g

Normalized fluorescence intensity versus time for Lucifer yellow and doxorubicin in microvessels formed from MDCK.2, MDCKII-w/t, or MDCKII-MDR1 cells. (a) Total fluorescence intensity (lumen + ECM) and (b) fluorescence intensity in the ECM following injection
Figure Legend Snippet: Normalized fluorescence intensity versus time for Lucifer yellow and doxorubicin in microvessels formed from MDCK.2, MDCKII-w/t, or MDCKII-MDR1 cells. (a) Total fluorescence intensity (lumen + ECM) and (b) fluorescence intensity in the ECM following injection

Techniques Used: Fluorescence, Injection

Doxorubicin and Lucifer yellow transport in microvessels. Overlays of phase contrast and fluorescence images of (a,b) MDCK.2 microvessels, (c,d) MDCKII-w/t microvessels, and (e,f) MDCKII-MDR1 microvessels on introduction of (a,c,e) Lucifer yellow and
Figure Legend Snippet: Doxorubicin and Lucifer yellow transport in microvessels. Overlays of phase contrast and fluorescence images of (a,b) MDCK.2 microvessels, (c,d) MDCKII-w/t microvessels, and (e,f) MDCKII-MDR1 microvessels on introduction of (a,c,e) Lucifer yellow and

Techniques Used: Fluorescence

Analysis of doxorubicin accumulation in cells following injection of 100 µM doxorubicin. (a) Fluorescence images of microvessels at each time point are used to obtain intensity profiles in the cells making up along vessel walls (white box) and
Figure Legend Snippet: Analysis of doxorubicin accumulation in cells following injection of 100 µM doxorubicin. (a) Fluorescence images of microvessels at each time point are used to obtain intensity profiles in the cells making up along vessel walls (white box) and

Techniques Used: Injection, Fluorescence

Permeability of doxorubicin and Lucifer yellow in microvessels formed from MDCK.2, MDCKII-w/t, or MDCKII-MDR1 cells. In all cases the permeability was calculated from the change in fluorescence intensity in the ECM. (a) Permeability for Lucifer yellow
Figure Legend Snippet: Permeability of doxorubicin and Lucifer yellow in microvessels formed from MDCK.2, MDCKII-w/t, or MDCKII-MDR1 cells. In all cases the permeability was calculated from the change in fluorescence intensity in the ECM. (a) Permeability for Lucifer yellow

Techniques Used: Permeability, Fluorescence

18) Product Images from "Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4+CD25+FoxP3+ regulatory T cells activation"

Article Title: Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4+CD25+FoxP3+ regulatory T cells activation

Journal: Theranostics

doi: 10.7150/thno.35045

Huh-7 macroH2A1 KD cells confer chemoresistance to parental cells in a paracrine manner. A. Nuclear fractions isolated from CTL HepG2 or Huh-7 cells, were processed for immunoblotting with anti-macroH2A1.1, anti-macroH2A1.2 and anti-H2B antibodies. B. Three experimental conditions: control (CTL), KD and CTL cells plus KD conditioned medium (CM). C. MTT assay in CTL, KD or CTL + CM cells incubated with or without vehicle (DMSO), 2 µM Doxorubicin (Doxo) or 1 µM Sorafenib for 72 h. Data represent the mean cell proliferation ± s.d. relative to CTL cells at 24 h. N=4. D. Population doubling time in CTL, KD or CTL + CM cells incubated with or without vehicle (DMSO), 2 µM Doxorubicin (Doxo) or 1 µM Sorafenib for 72 h. Data represent the mean cell proliferation ± s.d. N=3.*P
Figure Legend Snippet: Huh-7 macroH2A1 KD cells confer chemoresistance to parental cells in a paracrine manner. A. Nuclear fractions isolated from CTL HepG2 or Huh-7 cells, were processed for immunoblotting with anti-macroH2A1.1, anti-macroH2A1.2 and anti-H2B antibodies. B. Three experimental conditions: control (CTL), KD and CTL cells plus KD conditioned medium (CM). C. MTT assay in CTL, KD or CTL + CM cells incubated with or without vehicle (DMSO), 2 µM Doxorubicin (Doxo) or 1 µM Sorafenib for 72 h. Data represent the mean cell proliferation ± s.d. relative to CTL cells at 24 h. N=4. D. Population doubling time in CTL, KD or CTL + CM cells incubated with or without vehicle (DMSO), 2 µM Doxorubicin (Doxo) or 1 µM Sorafenib for 72 h. Data represent the mean cell proliferation ± s.d. N=3.*P

Techniques Used: Isolation, CTL Assay, MTT Assay, Incubation

Related Articles

Transduction:

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
Article Snippet: .. For the transduction of pHAEs, viral particles (BoV or AAV2 as control) were added to the apical side at MOIs ranging from 2 × 104 to 1 × 105 (as indicated for each experiment), and they were incubated overnight in the presence of two proteasome inhibitors, Doxorubicin (Santa Cruz Biotechnology, 5 μM) and Calpain inhibitor 1 ALLN (G-Biosciences, St. Louis, MO, USA; 786-057, 40 μM). .. For basolateral infection, the transwell inserts were flipped, and scAAV-Gluc/BoV was directly added to the surface of the filter at an MOI of 2 × 104 .

Centrifugation:

Article Title: Phosphorylation-Dependent Degradation of p300 by Doxorubicin-Activated p38 Mitogen-Activated Protein Kinase in Cardiac Cells
Article Snippet: Cardiomyocytes were also treated with 1 μM doxorubicin or cotreated with doxorubicin and 30 μM MG-132 for 21 h. After being labeled, the cells were extensively washed and lysed in RIPA buffer (Santa Cruz) supplemented with freshly made PPI and inhibitors (20 mM NaF, 20 mM β-glycerophosphate, 20 mM sodium orthovanadate, and 1 μM okadaic acid). .. After being washed, the agarose resin was recovered by centrifugation and resuspended in SDS-loading dye.

Luciferase:

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
Article Snippet: Transductions were always performed in the presence of 0.5–1 μM Doxorubicin (Santa Cruz Biotechnology, Dallas, TX, USA; 25316-40-9), and the viruses were left on the cells overnight. .. Transgene expression was assessed via fluorescence microscopy or Gaussia luciferase assay.

Positive Control:

Article Title: Anti-inflammatory and Antitumor Activity of a Triple Therapy for a Colitis-Related Colorectal Cancer
Article Snippet: Pharmacological treatments began after the second DSS cycle, and were administered daily during 40 days under the following scheme: Group 1: negative control, SSI (saline solution isotonic); Group 2: Doxorubicin 1 mg/Kg (positive control); Group 3: Metformin 200 mg/kg - Sodium Oxamate 15 mg/kg and Group 4: Metformin 200 mg/kg - Sodium Oxamate 15 mg/kg - Doxorubicin 1 mg/kg. .. Doxorubicin was obtained commercially as Doxolem RU, Metformin and Sodium Oxamate were purchased from Santa Cruz Biotechnology (sc-202000A and sc-215880A respectively).

Cytometry:

Article Title: The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival *The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival * χ
Article Snippet: The population of sub-G1 cells was analyzed by flow cytometry (UAB Flow Cytometry Core Facility). .. Similar procedures were performed in NIH 3T3 cells overexpressing a scrambled siRNA, an LPA2 siRNA, or a Siva-1 siRNA except that cells were pre-treated with 10 μ m LPA for 6 h followed by the addition of 10 μ m adriamycin for 20 h. Immunoblotting was performed to detect endogenous Siva-1, HA-Siva-1, procaspase-3, and β -actin using a Siva-1-specific polyclonal antibody, an anti-HA antibody (Santa Cruz Biotechnology), an anti-caspase-3 antibody (Santa Cruz Biotechnology), and an anti- β -actin antibody (Sigma), respectively.

Incubation:

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
Article Snippet: .. For the transduction of pHAEs, viral particles (BoV or AAV2 as control) were added to the apical side at MOIs ranging from 2 × 104 to 1 × 105 (as indicated for each experiment), and they were incubated overnight in the presence of two proteasome inhibitors, Doxorubicin (Santa Cruz Biotechnology, 5 μM) and Calpain inhibitor 1 ALLN (G-Biosciences, St. Louis, MO, USA; 786-057, 40 μM). .. For basolateral infection, the transwell inserts were flipped, and scAAV-Gluc/BoV was directly added to the surface of the filter at an MOI of 2 × 104 .

Article Title: The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival *The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival * χ
Article Snippet: NIH 3T3 cells transiently expressing HA-Siva-1 or not were incubated in 0.1% fatty acid-free BSA-containing Dulbecco’s modified Eagle’s medium without or with 5 μ m LPA for 6 h followed by the addition of 5 μ m adriamycin for another 24 h. The cells were fixed in 70% ethanol and DNA was stained with propidium iodide (Roche Applied Science). .. Similar procedures were performed in NIH 3T3 cells overexpressing a scrambled siRNA, an LPA2 siRNA, or a Siva-1 siRNA except that cells were pre-treated with 10 μ m LPA for 6 h followed by the addition of 10 μ m adriamycin for 20 h. Immunoblotting was performed to detect endogenous Siva-1, HA-Siva-1, procaspase-3, and β -actin using a Siva-1-specific polyclonal antibody, an anti-HA antibody (Santa Cruz Biotechnology), an anti-caspase-3 antibody (Santa Cruz Biotechnology), and an anti- β -actin antibody (Sigma), respectively.

Cell Culture:

Article Title: A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission
Article Snippet: 293FT cells (Invitrogen, R700–07) were cultured in DMEM containing 10% FBS, 0.5 mg/ml G418 (Sigma-Aldrich, A1720), 4 mM L-glutamine, 0.1 mM MEM nonessential amino acids (Gibco, 11140), 1 mM sodium pyruvate (Gibco, 11360). .. Liensinine (2586–96–1) was purchased from MUST bio-technology, 3-methyladenine (M9281), rapamycin (R117), acridine orange (158550), chloroquine diphosphate salt (C6628), Thiazolyl Blue Tetrazolium Blue (MTT, M2128), paclitaxel (T7191), vincristine (V8879) and cisplatin (P4394) were purchased from Sigma-Aldrich, bafilomycin A1 (11038) was purchased from Cayman Chemical, doxorubicin (sc-200923) and staurosporine (sc-360258) were purchased from Santa Cruz Biotechnology, and Mdivi-1 (s7162) was from Selleck Chemicals.

Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers
Article Snippet: Paragraph title: Cell culture ... Doxorubicin (sc-280681) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway
Article Snippet: .. After a 4-h exposure of cells to various doses of chemotherapeutic agents, Paclitaxel (sc-201439, Santa Cruz Biotechnology, Inc.) or Doxorubicin (sc-200923, Santa Cruz Biotechnology, Inc.), the cells were cultured for an additional 7 days with drug-free medium or medium with an PI3K/AKT pathway inhibitor LY294002 (#9901, Cell Signaling) or Perifosine (#14240, Cell Signaling), or an NF-κB pathway inhibitor Bay11-7082 (sc-200615, Santa Cruz Biotechnology, Inc.). .. Cells were then harvested by trypsinization at different time points and stained with trypan blue.

Article Title: Bcl-xL mediates therapeutic resistance of a mesenchymal breast cancer cell subpopulation
Article Snippet: Paragraph title: Cell culture, reagents and transfections ... Cisplatin (Neocorp), carboplatin (medac), doxorubicin (diluted in DMSO; Santa Cruz), neocarzinostatin (NCS), cycloheximide (CHX; diluted in Ethanol; both Sigma Aldrich), Trail, TNFalpha (both R & D systems), gossypol (SellectBio), Abt-737 (active biochem, both diluted in DMSO).

Expressing:

Article Title: A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission
Article Snippet: MDA-MB-231 cells stably expressing EGFP-LC3 were maintained in DMEM with 10% FBS and 200 μg/ml G418. .. Liensinine (2586–96–1) was purchased from MUST bio-technology, 3-methyladenine (M9281), rapamycin (R117), acridine orange (158550), chloroquine diphosphate salt (C6628), Thiazolyl Blue Tetrazolium Blue (MTT, M2128), paclitaxel (T7191), vincristine (V8879) and cisplatin (P4394) were purchased from Sigma-Aldrich, bafilomycin A1 (11038) was purchased from Cayman Chemical, doxorubicin (sc-200923) and staurosporine (sc-360258) were purchased from Santa Cruz Biotechnology, and Mdivi-1 (s7162) was from Selleck Chemicals.

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
Article Snippet: The same MOIs were used for the AAV2 controls carrying the same expression cassettes. .. Transductions were always performed in the presence of 0.5–1 μM Doxorubicin (Santa Cruz Biotechnology, Dallas, TX, USA; 25316-40-9), and the viruses were left on the cells overnight.

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
Article Snippet: Transgene expression was assessed via fluorescence microscopy or Gaussia luciferase assay. .. For the transduction of pHAEs, viral particles (BoV or AAV2 as control) were added to the apical side at MOIs ranging from 2 × 104 to 1 × 105 (as indicated for each experiment), and they were incubated overnight in the presence of two proteasome inhibitors, Doxorubicin (Santa Cruz Biotechnology, 5 μM) and Calpain inhibitor 1 ALLN (G-Biosciences, St. Louis, MO, USA; 786-057, 40 μM).

Article Title: MicroRNA-452 promotes stem-like cells of hepatocellular carcinoma by inhibiting Sox7 involving Wnt/β-catenin signaling pathway
Article Snippet: The Myc/CTNNB1, Myc/TCF4, Wnt1 expressing plasmid were purchased from Origene (USA). .. Doxorubicin and Sorafenib were purchased from Santa Cruz (USA).

Article Title: The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival *The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival * χ
Article Snippet: Paragraph title: Adriamycin-induced Siva-1 Expression, Caspase-3 Cleavage, and Apoptosis ... Similar procedures were performed in NIH 3T3 cells overexpressing a scrambled siRNA, an LPA2 siRNA, or a Siva-1 siRNA except that cells were pre-treated with 10 μ m LPA for 6 h followed by the addition of 10 μ m adriamycin for 20 h. Immunoblotting was performed to detect endogenous Siva-1, HA-Siva-1, procaspase-3, and β -actin using a Siva-1-specific polyclonal antibody, an anti-HA antibody (Santa Cruz Biotechnology), an anti-caspase-3 antibody (Santa Cruz Biotechnology), and an anti- β -actin antibody (Sigma), respectively.

Modification:

Article Title: A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission
Article Snippet: MDA-MB-231, MCF-7 and A549 cells were obtained from the American Type Culture Collection (ATCC, HTB-26, HTB-22, CCL-185) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, 10100–147) in 5% CO2 at 37°C, U937 cells (ATCC, CRL-1593.2) were cultured in RMPI 1640 (Gibco, 22400) with 10% FBS, LN229 cells (ATCC, CRL-2611) were cultured in DMEM/F-12 (Gibco, 10565) with 10% FBS. .. Liensinine (2586–96–1) was purchased from MUST bio-technology, 3-methyladenine (M9281), rapamycin (R117), acridine orange (158550), chloroquine diphosphate salt (C6628), Thiazolyl Blue Tetrazolium Blue (MTT, M2128), paclitaxel (T7191), vincristine (V8879) and cisplatin (P4394) were purchased from Sigma-Aldrich, bafilomycin A1 (11038) was purchased from Cayman Chemical, doxorubicin (sc-200923) and staurosporine (sc-360258) were purchased from Santa Cruz Biotechnology, and Mdivi-1 (s7162) was from Selleck Chemicals.

Article Title: The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival *The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival * χ
Article Snippet: NIH 3T3 cells transiently expressing HA-Siva-1 or not were incubated in 0.1% fatty acid-free BSA-containing Dulbecco’s modified Eagle’s medium without or with 5 μ m LPA for 6 h followed by the addition of 5 μ m adriamycin for another 24 h. The cells were fixed in 70% ethanol and DNA was stained with propidium iodide (Roche Applied Science). .. Similar procedures were performed in NIH 3T3 cells overexpressing a scrambled siRNA, an LPA2 siRNA, or a Siva-1 siRNA except that cells were pre-treated with 10 μ m LPA for 6 h followed by the addition of 10 μ m adriamycin for 20 h. Immunoblotting was performed to detect endogenous Siva-1, HA-Siva-1, procaspase-3, and β -actin using a Siva-1-specific polyclonal antibody, an anti-HA antibody (Santa Cruz Biotechnology), an anti-caspase-3 antibody (Santa Cruz Biotechnology), and an anti- β -actin antibody (Sigma), respectively.

Transfection:

Article Title: MicroRNA-452 promotes stem-like cells of hepatocellular carcinoma by inhibiting Sox7 involving Wnt/β-catenin signaling pathway
Article Snippet: Doxorubicin and Sorafenib were purchased from Santa Cruz (USA). .. Lipofectamine2000 was used for cell transfection according to the manufacturer's instructions.

Article Title: Bcl-xL mediates therapeutic resistance of a mesenchymal breast cancer cell subpopulation
Article Snippet: Paragraph title: Cell culture, reagents and transfections ... Cisplatin (Neocorp), carboplatin (medac), doxorubicin (diluted in DMSO; Santa Cruz), neocarzinostatin (NCS), cycloheximide (CHX; diluted in Ethanol; both Sigma Aldrich), Trail, TNFalpha (both R & D systems), gossypol (SellectBio), Abt-737 (active biochem, both diluted in DMSO).

Stable Transfection:

Article Title: A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission
Article Snippet: MDA-MB-231 cells stably expressing EGFP-LC3 were maintained in DMEM with 10% FBS and 200 μg/ml G418. .. Liensinine (2586–96–1) was purchased from MUST bio-technology, 3-methyladenine (M9281), rapamycin (R117), acridine orange (158550), chloroquine diphosphate salt (C6628), Thiazolyl Blue Tetrazolium Blue (MTT, M2128), paclitaxel (T7191), vincristine (V8879) and cisplatin (P4394) were purchased from Sigma-Aldrich, bafilomycin A1 (11038) was purchased from Cayman Chemical, doxorubicin (sc-200923) and staurosporine (sc-360258) were purchased from Santa Cruz Biotechnology, and Mdivi-1 (s7162) was from Selleck Chemicals.

Infection:

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
Article Snippet: For the transduction of pHAEs, viral particles (BoV or AAV2 as control) were added to the apical side at MOIs ranging from 2 × 104 to 1 × 105 (as indicated for each experiment), and they were incubated overnight in the presence of two proteasome inhibitors, Doxorubicin (Santa Cruz Biotechnology, 5 μM) and Calpain inhibitor 1 ALLN (G-Biosciences, St. Louis, MO, USA; 786-057, 40 μM). .. For basolateral infection, the transwell inserts were flipped, and scAAV-Gluc/BoV was directly added to the surface of the filter at an MOI of 2 × 104 .

Clonogenic Cell Survival Assay:

Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway
Article Snippet: Paragraph title: Survival assay and cell counting ... After a 4-h exposure of cells to various doses of chemotherapeutic agents, Paclitaxel (sc-201439, Santa Cruz Biotechnology, Inc.) or Doxorubicin (sc-200923, Santa Cruz Biotechnology, Inc.), the cells were cultured for an additional 7 days with drug-free medium or medium with an PI3K/AKT pathway inhibitor LY294002 (#9901, Cell Signaling) or Perifosine (#14240, Cell Signaling), or an NF-κB pathway inhibitor Bay11-7082 (sc-200615, Santa Cruz Biotechnology, Inc.).

Inverted Microscopy:

Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway
Article Snippet: After a 4-h exposure of cells to various doses of chemotherapeutic agents, Paclitaxel (sc-201439, Santa Cruz Biotechnology, Inc.) or Doxorubicin (sc-200923, Santa Cruz Biotechnology, Inc.), the cells were cultured for an additional 7 days with drug-free medium or medium with an PI3K/AKT pathway inhibitor LY294002 (#9901, Cell Signaling) or Perifosine (#14240, Cell Signaling), or an NF-κB pathway inhibitor Bay11-7082 (sc-200615, Santa Cruz Biotechnology, Inc.). .. Viable cells were counted using a hematocytometer and an inverted microscope.

Recombinant:

Article Title: FLASH Knockdown Sensitizes Cells To Fas-Mediated Apoptosis via Down-Regulation of the Anti-Apoptotic Proteins, MCL-1 and Cflip Short
Article Snippet: A panel of caspase inhibitors (FMKSP01) and recombinant human Fas Ligand/TNF9SF (126-FL-010) were purchased from R & D (Minneapolis, MN). .. MG132, cycloheximide (CHX), actinomycin D were from Sigma and adriamycin was from Santa Cruz.

MTT Assay:

Article Title: A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission
Article Snippet: .. Liensinine (2586–96–1) was purchased from MUST bio-technology, 3-methyladenine (M9281), rapamycin (R117), acridine orange (158550), chloroquine diphosphate salt (C6628), Thiazolyl Blue Tetrazolium Blue (MTT, M2128), paclitaxel (T7191), vincristine (V8879) and cisplatin (P4394) were purchased from Sigma-Aldrich, bafilomycin A1 (11038) was purchased from Cayman Chemical, doxorubicin (sc-200923) and staurosporine (sc-360258) were purchased from Santa Cruz Biotechnology, and Mdivi-1 (s7162) was from Selleck Chemicals. ..

In Vivo:

Article Title: Anti-inflammatory and Antitumor Activity of a Triple Therapy for a Colitis-Related Colorectal Cancer
Article Snippet: Paragraph title: In vivo studies ... Doxorubicin was obtained commercially as Doxolem RU, Metformin and Sodium Oxamate were purchased from Santa Cruz Biotechnology (sc-202000A and sc-215880A respectively).

Fluorescence:

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
Article Snippet: Transductions were always performed in the presence of 0.5–1 μM Doxorubicin (Santa Cruz Biotechnology, Dallas, TX, USA; 25316-40-9), and the viruses were left on the cells overnight. .. Transgene expression was assessed via fluorescence microscopy or Gaussia luciferase assay.

Article Title: The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival *The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival * χ
Article Snippet: Similar procedures were performed in NIH 3T3 cells overexpressing a scrambled siRNA, an LPA2 siRNA, or a Siva-1 siRNA except that cells were pre-treated with 10 μ m LPA for 6 h followed by the addition of 10 μ m adriamycin for 20 h. Immunoblotting was performed to detect endogenous Siva-1, HA-Siva-1, procaspase-3, and β -actin using a Siva-1-specific polyclonal antibody, an anti-HA antibody (Santa Cruz Biotechnology), an anti-caspase-3 antibody (Santa Cruz Biotechnology), and an anti- β -actin antibody (Sigma), respectively. .. Apoptosis was also determined by annexin V-fluorescein isothiocyanate staining (BD Biosciences) followed by fluorescence-activated cell sorter analysis.

Multiple Displacement Amplification:

Article Title: A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission
Article Snippet: MDA-MB-231 cells with a stable knockdown of ATG5 or ATG7 were maintained in DMEM with 10% FBS and 15 μg/ml puromycin (Sigma-Aldrich, P9620). .. Liensinine (2586–96–1) was purchased from MUST bio-technology, 3-methyladenine (M9281), rapamycin (R117), acridine orange (158550), chloroquine diphosphate salt (C6628), Thiazolyl Blue Tetrazolium Blue (MTT, M2128), paclitaxel (T7191), vincristine (V8879) and cisplatin (P4394) were purchased from Sigma-Aldrich, bafilomycin A1 (11038) was purchased from Cayman Chemical, doxorubicin (sc-200923) and staurosporine (sc-360258) were purchased from Santa Cruz Biotechnology, and Mdivi-1 (s7162) was from Selleck Chemicals.

Flow Cytometry:

Article Title: The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival *The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival * χ
Article Snippet: The population of sub-G1 cells was analyzed by flow cytometry (UAB Flow Cytometry Core Facility). .. Similar procedures were performed in NIH 3T3 cells overexpressing a scrambled siRNA, an LPA2 siRNA, or a Siva-1 siRNA except that cells were pre-treated with 10 μ m LPA for 6 h followed by the addition of 10 μ m adriamycin for 20 h. Immunoblotting was performed to detect endogenous Siva-1, HA-Siva-1, procaspase-3, and β -actin using a Siva-1-specific polyclonal antibody, an anti-HA antibody (Santa Cruz Biotechnology), an anti-caspase-3 antibody (Santa Cruz Biotechnology), and an anti- β -actin antibody (Sigma), respectively.

Labeling:

Article Title: Phosphorylation-Dependent Degradation of p300 by Doxorubicin-Activated p38 Mitogen-Activated Protein Kinase in Cardiac Cells
Article Snippet: .. Cardiomyocytes were also treated with 1 μM doxorubicin or cotreated with doxorubicin and 30 μM MG-132 for 21 h. After being labeled, the cells were extensively washed and lysed in RIPA buffer (Santa Cruz) supplemented with freshly made PPI and inhibitors (20 mM NaF, 20 mM β-glycerophosphate, 20 mM sodium orthovanadate, and 1 μM okadaic acid). .. Endogenous p300 was immunoprecipitated with an anti-p300 antibody (N-15), and was preadsorbed on protein A/G PLUS agarose.

Microscopy:

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
Article Snippet: Transductions were always performed in the presence of 0.5–1 μM Doxorubicin (Santa Cruz Biotechnology, Dallas, TX, USA; 25316-40-9), and the viruses were left on the cells overnight. .. Transgene expression was assessed via fluorescence microscopy or Gaussia luciferase assay.

Plasmid Preparation:

Article Title: MicroRNA-452 promotes stem-like cells of hepatocellular carcinoma by inhibiting Sox7 involving Wnt/β-catenin signaling pathway
Article Snippet: The TCF Reporter Plasmid Kit were brought from Millipore (USA). .. Doxorubicin and Sorafenib were purchased from Santa Cruz (USA).

Negative Control:

Article Title: Anti-inflammatory and Antitumor Activity of a Triple Therapy for a Colitis-Related Colorectal Cancer
Article Snippet: Pharmacological treatments began after the second DSS cycle, and were administered daily during 40 days under the following scheme: Group 1: negative control, SSI (saline solution isotonic); Group 2: Doxorubicin 1 mg/Kg (positive control); Group 3: Metformin 200 mg/kg - Sodium Oxamate 15 mg/kg and Group 4: Metformin 200 mg/kg - Sodium Oxamate 15 mg/kg - Doxorubicin 1 mg/kg. .. Doxorubicin was obtained commercially as Doxolem RU, Metformin and Sodium Oxamate were purchased from Santa Cruz Biotechnology (sc-202000A and sc-215880A respectively).

Immunoprecipitation:

Article Title: Phosphorylation-Dependent Degradation of p300 by Doxorubicin-Activated p38 Mitogen-Activated Protein Kinase in Cardiac Cells
Article Snippet: Paragraph title: Metabolic labeling and immunoprecipitation. ... Cardiomyocytes were also treated with 1 μM doxorubicin or cotreated with doxorubicin and 30 μM MG-132 for 21 h. After being labeled, the cells were extensively washed and lysed in RIPA buffer (Santa Cruz) supplemented with freshly made PPI and inhibitors (20 mM NaF, 20 mM β-glycerophosphate, 20 mM sodium orthovanadate, and 1 μM okadaic acid).

Cell Counting:

Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway
Article Snippet: Paragraph title: Survival assay and cell counting ... After a 4-h exposure of cells to various doses of chemotherapeutic agents, Paclitaxel (sc-201439, Santa Cruz Biotechnology, Inc.) or Doxorubicin (sc-200923, Santa Cruz Biotechnology, Inc.), the cells were cultured for an additional 7 days with drug-free medium or medium with an PI3K/AKT pathway inhibitor LY294002 (#9901, Cell Signaling) or Perifosine (#14240, Cell Signaling), or an NF-κB pathway inhibitor Bay11-7082 (sc-200615, Santa Cruz Biotechnology, Inc.).

Staining:

Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway
Article Snippet: After a 4-h exposure of cells to various doses of chemotherapeutic agents, Paclitaxel (sc-201439, Santa Cruz Biotechnology, Inc.) or Doxorubicin (sc-200923, Santa Cruz Biotechnology, Inc.), the cells were cultured for an additional 7 days with drug-free medium or medium with an PI3K/AKT pathway inhibitor LY294002 (#9901, Cell Signaling) or Perifosine (#14240, Cell Signaling), or an NF-κB pathway inhibitor Bay11-7082 (sc-200615, Santa Cruz Biotechnology, Inc.). .. Cells were then harvested by trypsinization at different time points and stained with trypan blue.

Article Title: The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival *The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival * χ
Article Snippet: NIH 3T3 cells transiently expressing HA-Siva-1 or not were incubated in 0.1% fatty acid-free BSA-containing Dulbecco’s modified Eagle’s medium without or with 5 μ m LPA for 6 h followed by the addition of 5 μ m adriamycin for another 24 h. The cells were fixed in 70% ethanol and DNA was stained with propidium iodide (Roche Applied Science). .. Similar procedures were performed in NIH 3T3 cells overexpressing a scrambled siRNA, an LPA2 siRNA, or a Siva-1 siRNA except that cells were pre-treated with 10 μ m LPA for 6 h followed by the addition of 10 μ m adriamycin for 20 h. Immunoblotting was performed to detect endogenous Siva-1, HA-Siva-1, procaspase-3, and β -actin using a Siva-1-specific polyclonal antibody, an anti-HA antibody (Santa Cruz Biotechnology), an anti-caspase-3 antibody (Santa Cruz Biotechnology), and an anti- β -actin antibody (Sigma), respectively.

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    Santa Cruz Biotechnology doxorubicin
    Uev1A promotes AKT-mediated chemoresistance in breast cancer cells. a , b Effects of UEV1 overexpression on chemoresistance of MDA-MB-231 cells. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. After a 4-h exposure to various doses of chemotherapeutic agents Paclitaxel ( a ) or <t>Doxorubicin</t> ( b ), the cells were cultured for an additional 7 days with drug-free medium containing 10% FBS. Then cells were harvested by trypsinization and stained with trypan blue. Cell viability was assayed by cell number counting using a hematocytometer and an inverted microscope. Each sample was measured in triplicate and repeated 2 times. c , d Dependence of chemoresistance of MDA-MB-231 cells to the AKT pathway. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. With 10 μM LY294002 pretreated for 12 h, cells were exposed to various doses of Paclitaxel ( c ) or Doxorubicin ( d ) which were also in the medium with 10 µM LY294002 for 4 h and then cultured for an additional 7 days with medium containing 10% FBS and 10 µM LY294002. Cell viability assay was as described in a , b . e , f Effects of UEV1 overexpression on chemoresistance of MCF7 cells. Experimental conditions were as described in a , b . g , h Dependence of chemoresistance of MCF7 cells to the AKT pathway. Experimental conditions were as described in c , d . * P
    Doxorubicin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology drugs doxorubicin doxolem
    Cytotoxic effect of Metformin, Oxamate or <t>Doxorubicin,</t> and their combinations against MDA-MB-231 cells in the SRB assay. MDA-MB-231 cells were treated with Met, Ox, or Dox alone, and all possible combinations for 4, 24, 48 and 72 hours at different concentrations. Estimated IC 50 values are Dox 0.5 μM, Met 25 mM, and Ox 15 mM.
    Drugs Doxorubicin Doxolem, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology myocyte enhancer factor 2c
    Multipotent capacity of cardiac stem cells (A) Cardiac and smooth muscle differentiation of CSCs in vitro . Immunostaining of GFP positive CSCs with cardiac troponin T (cTnT), myocyte enhancer <t>factor</t> 2C (MEF-2C), connexin-43, and α-smooth muscle actin (α-SMA). (B) Endothelial differentiation of CSCs in vitro . The cells were cultured in EGM-2 medium with 10 ng/ml VEGF and showed endothelial differentiation by morphology (I) and uptake of Dil-ac-LDL (II) . Endothelial tube formation by differentiated CSCs after 12 hours of plating on Matrigel (III ) whereas undifferentiated CSCs cells do not form cord-like structures (IV) . ( C) Oil red staining and RT-PCR analysis of PPARγ and lipoprotein lipase (LPL) expression shows adipogenic differentiation of the CSCs induced for 2 weeks. (D) Alizarin red S staining of calcium and RT-PCR analysis of osteocalcin expression shows CSCs induced to differentiate into osteoblasts.
    Myocyte Enhancer Factor 2c, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Uev1A promotes AKT-mediated chemoresistance in breast cancer cells. a , b Effects of UEV1 overexpression on chemoresistance of MDA-MB-231 cells. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. After a 4-h exposure to various doses of chemotherapeutic agents Paclitaxel ( a ) or Doxorubicin ( b ), the cells were cultured for an additional 7 days with drug-free medium containing 10% FBS. Then cells were harvested by trypsinization and stained with trypan blue. Cell viability was assayed by cell number counting using a hematocytometer and an inverted microscope. Each sample was measured in triplicate and repeated 2 times. c , d Dependence of chemoresistance of MDA-MB-231 cells to the AKT pathway. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. With 10 μM LY294002 pretreated for 12 h, cells were exposed to various doses of Paclitaxel ( c ) or Doxorubicin ( d ) which were also in the medium with 10 µM LY294002 for 4 h and then cultured for an additional 7 days with medium containing 10% FBS and 10 µM LY294002. Cell viability assay was as described in a , b . e , f Effects of UEV1 overexpression on chemoresistance of MCF7 cells. Experimental conditions were as described in a , b . g , h Dependence of chemoresistance of MCF7 cells to the AKT pathway. Experimental conditions were as described in c , d . * P

    Journal: Cancer Cell International

    Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway

    doi: 10.1186/s12935-019-1050-4

    Figure Lengend Snippet: Uev1A promotes AKT-mediated chemoresistance in breast cancer cells. a , b Effects of UEV1 overexpression on chemoresistance of MDA-MB-231 cells. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. After a 4-h exposure to various doses of chemotherapeutic agents Paclitaxel ( a ) or Doxorubicin ( b ), the cells were cultured for an additional 7 days with drug-free medium containing 10% FBS. Then cells were harvested by trypsinization and stained with trypan blue. Cell viability was assayed by cell number counting using a hematocytometer and an inverted microscope. Each sample was measured in triplicate and repeated 2 times. c , d Dependence of chemoresistance of MDA-MB-231 cells to the AKT pathway. UEV1A -overexpressed or vector control MDA-MB-231-TR cells were seeded onto 6-well culture plates with doxycycline. With 10 μM LY294002 pretreated for 12 h, cells were exposed to various doses of Paclitaxel ( c ) or Doxorubicin ( d ) which were also in the medium with 10 µM LY294002 for 4 h and then cultured for an additional 7 days with medium containing 10% FBS and 10 µM LY294002. Cell viability assay was as described in a , b . e , f Effects of UEV1 overexpression on chemoresistance of MCF7 cells. Experimental conditions were as described in a , b . g , h Dependence of chemoresistance of MCF7 cells to the AKT pathway. Experimental conditions were as described in c , d . * P

    Article Snippet: After a 4-h exposure of cells to various doses of chemotherapeutic agents, Paclitaxel (sc-201439, Santa Cruz Biotechnology, Inc.) or Doxorubicin (sc-200923, Santa Cruz Biotechnology, Inc.), the cells were cultured for an additional 7 days with drug-free medium or medium with an PI3K/AKT pathway inhibitor LY294002 (#9901, Cell Signaling) or Perifosine (#14240, Cell Signaling), or an NF-κB pathway inhibitor Bay11-7082 (sc-200615, Santa Cruz Biotechnology, Inc.).

    Techniques: Over Expression, Multiple Displacement Amplification, Plasmid Preparation, Cell Culture, Staining, Inverted Microscopy, Viability Assay

    Uev1A inhibits apoptosis through the AKT pathway in breast cancer cells. a , b UEV1A -overexpressed or vector control MDA-MB-231-TR cells with Paclitaxel (Pacl) ( a ) or Doxorubicin (Doxo) ( b ). c , d UEV1A -overexpressed or vector control MCF7 cells with Paclitaxel (Pacl) ( c ) or Doxorubicin (Doxo) ( d ). Cells were pretreated with (right panel) or without (left panel) 10 µM LY294002 (LY) for 12 h and then exposed to Paclitaxel or Doxorubicin, harvested at different time points and the protein levels of total PARP and cleaved-PARP (C-PARP) were detected by western blot. The ectopic Uev1A was monitored by an anti-HA antibody

    Journal: Cancer Cell International

    Article Title: Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway

    doi: 10.1186/s12935-019-1050-4

    Figure Lengend Snippet: Uev1A inhibits apoptosis through the AKT pathway in breast cancer cells. a , b UEV1A -overexpressed or vector control MDA-MB-231-TR cells with Paclitaxel (Pacl) ( a ) or Doxorubicin (Doxo) ( b ). c , d UEV1A -overexpressed or vector control MCF7 cells with Paclitaxel (Pacl) ( c ) or Doxorubicin (Doxo) ( d ). Cells were pretreated with (right panel) or without (left panel) 10 µM LY294002 (LY) for 12 h and then exposed to Paclitaxel or Doxorubicin, harvested at different time points and the protein levels of total PARP and cleaved-PARP (C-PARP) were detected by western blot. The ectopic Uev1A was monitored by an anti-HA antibody

    Article Snippet: After a 4-h exposure of cells to various doses of chemotherapeutic agents, Paclitaxel (sc-201439, Santa Cruz Biotechnology, Inc.) or Doxorubicin (sc-200923, Santa Cruz Biotechnology, Inc.), the cells were cultured for an additional 7 days with drug-free medium or medium with an PI3K/AKT pathway inhibitor LY294002 (#9901, Cell Signaling) or Perifosine (#14240, Cell Signaling), or an NF-κB pathway inhibitor Bay11-7082 (sc-200615, Santa Cruz Biotechnology, Inc.).

    Techniques: Plasmid Preparation, Multiple Displacement Amplification, Western Blot

    Downregulation of HES1 expression could inhibit Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 shRNA plasmids. Cells transfected with empty pLko.1 vector were used as the control group. ( A ) The knockdown efficiency of HES1 was measured by real-time RT-PCR. ( B ) The cell viability of knockdown cell lines was detected by MTT. ( C ) The knockdown and control cells were treated with Doxorubicin (300 μM) for 24 hours and then stained with Multicaspase/7-AAD for the quantitative measurement by using flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of ( C ). ** P

    Journal: Cancer Management and Research

    Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers

    doi: 10.2147/CMAR.S160315

    Figure Lengend Snippet: Downregulation of HES1 expression could inhibit Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 shRNA plasmids. Cells transfected with empty pLko.1 vector were used as the control group. ( A ) The knockdown efficiency of HES1 was measured by real-time RT-PCR. ( B ) The cell viability of knockdown cell lines was detected by MTT. ( C ) The knockdown and control cells were treated with Doxorubicin (300 μM) for 24 hours and then stained with Multicaspase/7-AAD for the quantitative measurement by using flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of ( C ). ** P

    Article Snippet: Doxorubicin (sc-280681) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Expressing, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Quantitative RT-PCR, MTT Assay, Staining, Flow Cytometry, Cytometry

    HES1 interplays with PARP1 to regulate AIF subcellular location. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Immunofluorescence of AIF (green) in the HES1-knockdown and control cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. ( B ) Immunofluorescence of AIF (green) in the HES1-overexpressing and control cells. The picture shows the location of AIF (green) in cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. Abbreviations: AIF, apoptosis-inducing factor; PARP1, poly(ADP-ribose) polymerase-1.

    Journal: Cancer Management and Research

    Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers

    doi: 10.2147/CMAR.S160315

    Figure Lengend Snippet: HES1 interplays with PARP1 to regulate AIF subcellular location. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Immunofluorescence of AIF (green) in the HES1-knockdown and control cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. ( B ) Immunofluorescence of AIF (green) in the HES1-overexpressing and control cells. The picture shows the location of AIF (green) in cells. DAPI staining (blue) marks the nuclei. Merged images are also shown. Abbreviations: AIF, apoptosis-inducing factor; PARP1, poly(ADP-ribose) polymerase-1.

    Article Snippet: Doxorubicin (sc-280681) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Immunofluorescence, Staining

    HES1 regulates apoptosis by activating PARP1. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Cell apoptosis of HES1-knockdown cell lines was determined by flow cytometry. ( B ) Bars represent the mean of three independent experiments performed in triplicate of ( A ). ( C ) Cell apoptosis of HES1 overexpressing cell line was determined by flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of (C). ** P

    Journal: Cancer Management and Research

    Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers

    doi: 10.2147/CMAR.S160315

    Figure Lengend Snippet: HES1 regulates apoptosis by activating PARP1. Notes: HeLa cells were pretreated with PARP1 inhibitor Rucaparib (1 nM) for 7 days, and then, the cells were treated with Doxorubicin (300 μM) for 24 hours. ( A ) Cell apoptosis of HES1-knockdown cell lines was determined by flow cytometry. ( B ) Bars represent the mean of three independent experiments performed in triplicate of ( A ). ( C ) Cell apoptosis of HES1 overexpressing cell line was determined by flow cytometry. ( D ) Bars represent the mean of three independent experiments performed in triplicate of (C). ** P

    Article Snippet: Doxorubicin (sc-280681) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Flow Cytometry, Cytometry

    Upregulation of HES1 expression could promote Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 overexpression plasmids. Cells transfected with empty pOZ vector were used as the control group. ( A ) The overexpression efficiency of HES1 was measured by real-time RT-PCR. ( B ) The levels of the overexpressed protein were detected by Western blot. ( C ) The cell viability of overexpressed cell line was detected by MTT. ( D ) The overexpressed cells were stained with Multicaspase/7-AAD for quantitative measurement by using flow cytometry. ( E ) Bars represent the mean of three independent experiments performed in triplicate of ( D ). # P > 0.05, ** P

    Journal: Cancer Management and Research

    Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers

    doi: 10.2147/CMAR.S160315

    Figure Lengend Snippet: Upregulation of HES1 expression could promote Doxorubicin-induced apoptosis. Notes: Cells were stably transfected with HES1 overexpression plasmids. Cells transfected with empty pOZ vector were used as the control group. ( A ) The overexpression efficiency of HES1 was measured by real-time RT-PCR. ( B ) The levels of the overexpressed protein were detected by Western blot. ( C ) The cell viability of overexpressed cell line was detected by MTT. ( D ) The overexpressed cells were stained with Multicaspase/7-AAD for quantitative measurement by using flow cytometry. ( E ) Bars represent the mean of three independent experiments performed in triplicate of ( D ). # P > 0.05, ** P

    Article Snippet: Doxorubicin (sc-280681) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Expressing, Stable Transfection, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, MTT Assay, Staining, Flow Cytometry, Cytometry

    Doxorubicin affects Hela S3 cell apoptosis. Notes: The HeLa S3 cells were treated with Doxorubicin (0, 100, 300, 600, 900 and 1200 μM) for 24 hours. ( A ) The percentage of apoptosis cells is shown. ( B ) Bars represent mean of three independent experiments performed in triplicate.

    Journal: Cancer Management and Research

    Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers

    doi: 10.2147/CMAR.S160315

    Figure Lengend Snippet: Doxorubicin affects Hela S3 cell apoptosis. Notes: The HeLa S3 cells were treated with Doxorubicin (0, 100, 300, 600, 900 and 1200 μM) for 24 hours. ( A ) The percentage of apoptosis cells is shown. ( B ) Bars represent mean of three independent experiments performed in triplicate.

    Article Snippet: Doxorubicin (sc-280681) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques:

    Effects of different concentrations of Doxorubicin on the mRNA expression of Notch pathway members. Notes: ( A ) HeLa S3 cells were treated with Doxorubicin (0, 300, 900 and 1200 μM) for 24 hours. The mRNA expression levels of Notch pathway components and targets were examined by real-time RT-PCR. ( B ) Selective representation of Doxorubicin-responsive Notch pathway genes. GAPDH was used as an internal control for the normalization of gene expression. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.

    Journal: Cancer Management and Research

    Article Title: Notch signaling pathway mediates Doxorubicin-driven apoptosis in cancers

    doi: 10.2147/CMAR.S160315

    Figure Lengend Snippet: Effects of different concentrations of Doxorubicin on the mRNA expression of Notch pathway members. Notes: ( A ) HeLa S3 cells were treated with Doxorubicin (0, 300, 900 and 1200 μM) for 24 hours. The mRNA expression levels of Notch pathway components and targets were examined by real-time RT-PCR. ( B ) Selective representation of Doxorubicin-responsive Notch pathway genes. GAPDH was used as an internal control for the normalization of gene expression. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.

    Article Snippet: Doxorubicin (sc-280681) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Cytotoxic effect of Metformin, Oxamate or Doxorubicin, and their combinations against MDA-MB-231 cells in the SRB assay. MDA-MB-231 cells were treated with Met, Ox, or Dox alone, and all possible combinations for 4, 24, 48 and 72 hours at different concentrations. Estimated IC 50 values are Dox 0.5 μM, Met 25 mM, and Ox 15 mM.

    Journal: Journal of Cancer

    Article Title: Targeting Metabolic Remodeling in Triple Negative Breast Cancer in a Murine Model

    doi: 10.7150/jca.16387

    Figure Lengend Snippet: Cytotoxic effect of Metformin, Oxamate or Doxorubicin, and their combinations against MDA-MB-231 cells in the SRB assay. MDA-MB-231 cells were treated with Met, Ox, or Dox alone, and all possible combinations for 4, 24, 48 and 72 hours at different concentrations. Estimated IC 50 values are Dox 0.5 μM, Met 25 mM, and Ox 15 mM.

    Article Snippet: Drugs Doxorubicin (Doxolem) (Dox); Metformin (1-1dimethylbiguanidine hydrochloride, Santa Cruz® sc-202000A) (Met); Sodium Oxamate (Ox) (Santa Cruz sc-215880).

    Techniques: Multiple Displacement Amplification, Sulforhodamine B Assay

    Multipotent capacity of cardiac stem cells (A) Cardiac and smooth muscle differentiation of CSCs in vitro . Immunostaining of GFP positive CSCs with cardiac troponin T (cTnT), myocyte enhancer factor 2C (MEF-2C), connexin-43, and α-smooth muscle actin (α-SMA). (B) Endothelial differentiation of CSCs in vitro . The cells were cultured in EGM-2 medium with 10 ng/ml VEGF and showed endothelial differentiation by morphology (I) and uptake of Dil-ac-LDL (II) . Endothelial tube formation by differentiated CSCs after 12 hours of plating on Matrigel (III ) whereas undifferentiated CSCs cells do not form cord-like structures (IV) . ( C) Oil red staining and RT-PCR analysis of PPARγ and lipoprotein lipase (LPL) expression shows adipogenic differentiation of the CSCs induced for 2 weeks. (D) Alizarin red S staining of calcium and RT-PCR analysis of osteocalcin expression shows CSCs induced to differentiate into osteoblasts.

    Journal: Journal of the American College of Cardiology

    Article Title: Imaging Survival and Function of Transplanted Cardiac Resident Stem Cells

    doi: 10.1016/j.jacc.2008.12.036

    Figure Lengend Snippet: Multipotent capacity of cardiac stem cells (A) Cardiac and smooth muscle differentiation of CSCs in vitro . Immunostaining of GFP positive CSCs with cardiac troponin T (cTnT), myocyte enhancer factor 2C (MEF-2C), connexin-43, and α-smooth muscle actin (α-SMA). (B) Endothelial differentiation of CSCs in vitro . The cells were cultured in EGM-2 medium with 10 ng/ml VEGF and showed endothelial differentiation by morphology (I) and uptake of Dil-ac-LDL (II) . Endothelial tube formation by differentiated CSCs after 12 hours of plating on Matrigel (III ) whereas undifferentiated CSCs cells do not form cord-like structures (IV) . ( C) Oil red staining and RT-PCR analysis of PPARγ and lipoprotein lipase (LPL) expression shows adipogenic differentiation of the CSCs induced for 2 weeks. (D) Alizarin red S staining of calcium and RT-PCR analysis of osteocalcin expression shows CSCs induced to differentiate into osteoblasts.

    Article Snippet: After 1 week, the cells were fixed with 4% paraformaldehyde and processed for immunofluorescence with antibodies specific to cardiac troponin T (cTnT), myocyte enhancer factor 2C (MEF-2C), connexin 43, and α-smooth muscle actin (SMA) (all from Santa Cruz Biotech, Santa Cruz, CA) in situ.

    Techniques: In Vitro, Immunostaining, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing